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TABLE OF CONTENTS
PRODUCT DESCRIPTION
SHIPPING CONDITIONS
STORAGE CONDITIONS
STABILITY
QC SPECIFICATIONS
PROTOCOL & APPLICATION NOTES
Modification/scale/purification
Manufacturing Details
Fluorescein
Rhodamine
HEX/TET/FAM
Phosphate
Biotin
Amine
Gateway
Alexa
Other dyes
Alkaline Phosphatase
Horse Radish Peroxidase
Phosphorothioate
“Fully-Phosphorothioated" Custom Primers
Aldehyde
Acridine
Thiol
Delivery Schedule
OligoPerfect Designed Primers
3' Modifications of Oligos
Reconstitution Protocol
A260/A280 ratio of the oligo
Oligo Visualization
Troubleshooting
“Custom Custom” modifications
List of current technology limitations
COMPETITOR INFORMATION
ALTERNATE PRODUCTS & COMPATIBILITY
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBER
COMPONENTS
DISCONTINUATION NOTICE
ASSOCIATED PRODUCTS
PRODUCT QUALITY ISSUES
Returning Primers
LICENSING
INTERNAL CONTACTS
PRODUCT DESCRIPTION
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Custom DNA Primers are synthetic oligonucleotides with specified sequence made to order for use in a variety of applications from PCR and sequencing to probes for gene detection. Custom DNA Primers are available as standard deoxynucleotides, modified bases, 5′ modified nucleotides (including fluorescent dyes, enzyme conjugates), and S-oligos for antisense studies. Five scales and four purity levels are available.
A unique and proprietary technology and an optimized production process is used to deliver quality Custom Primers, quickly and efficiently. The process includes easy ordering, linkage to integrated workstations for guaranteed accuracy, high-throughput synthesis, monitoring by a series of stringent quality control steps, useful product packaging, and overnight shipping for rapid, reliable delivery. Custom oligonucleotides are synthesized on automated, proprietary DNA synthesizers using standard cyanoethyl phosphoramidite chemistry. Consistent oligonucleotide quality is ensured by in-process trityl monitoring and quality control analysis. A comprehensive Certificate of Analysis accompanies each order, indicating quantity, molecular weight, extinction coefficient, sequence, and melting temperature.
DNA oligos can be ordered in tubes or plates:
?Tubes:
o Standard tube with cap (Scientific Specialties Inc. (SSI) 2680-00 (tube), 2001-00 (cap)). Volume is 2000μl ?Plates:
o96-wells:
PCR shallow 96-well plate (Abgene AB-0800): 200μl max volume, 180μl working volume; plate code 96N – 180. Shape of wells: V
Standard shallow 96-well plate (Corning Costar 3359): 360μl max volume, 250μl working volume;
plate code 96B – 250. Shape of wells: U/round. OLD SHALLOW PLATE
Standard medium 96-well plate (Abgene AB-0765): 800μl max volume, 650μl working volume; plate code 96L – 650. Shape of well: conical.
Cluster tube screwtop 96-well plate (Matrix ScrewTop TrackMates 3742, https://www.doczj.com/doc/0c17643869.html,): 1000μl max volume, 750μl working volume; plate code 96D – 750. Shape of well: Unknown.
SPECIAL I
Standard deep 96-well plate (Thompson Instruments, Oceanside distributor, part number 951651B): 1200μl max volume, 800μl working volume (equivalent to Abgene AB-0564); plate code 96C – 800.
Shape of well: U/round. OLD DEEP WELL PLATE
Cluster tube capmat 96-well plate (Matrix TrakMates 3791, https://www.doczj.com/doc/0c17643869.html,): 1400μl max volume, 850μl working volume; plate code 96J – 850. Shape of well: V. SPECIAL II o384 wells:
Standard 384-well plate – Thompson Instruments (Oceanside distributor) part number 931504B, 120μl max volume, 75μl working volume (equivalent to Abgene AB-0781); plate code 384E – 75. Shape of
well: pyramidal.
Microarray 384-well plate (Genetix X7020): 65μl max volume, 45μl working volume; plate code 384F – 45. Shape of well: flat.
o Seals and caps:
Heat Seal – Thompson Instruments (Oceanside distributor) part number 899406 (equivalent to Abgene AB-0745)
Adhesive Seal – Thompson Instruments (Oceanside distributor) part number 899405-1
Caps – Matrix ScrewTop caps 4472, for use with cluster tube screwtop 96-well plate, 96 caps required for each plate
Capmat – Matrix SepraSeal Cap Mats 4463, for use with cluster tube capmat 96-well plate, one mat required for each plate
SHIPPING CONDITIONS
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Please refer to Delivery Schedule.
Primary
Room Temperature lyophilized for tubes, different options for plates (lyophilized, ambient in solution, frozen in dry ice. Secondary
Special Handling by request: DNA oligos ordered in plates can be requested to a variety of normalization, pooling and aliquoting options free of charge. For TUBE oligos, the standard format is dried down and ship at room temperature. However, oligos ordered in tubes can be requested to be sent with a few pre-selected special handling options. These options are available in the check out section of the online cart. Two of these options can be checked for an extra $1 charge per primer. The options are re-suspension of the oligos in water to a certain concentration (10, 20, 50, 100 or 200 μM), electronic COA, second set of labels, 100% QC (for DNA oligos we only do Mass Spec or CE for 25% of the oligos, this options will make the oligos go through CE/Mass Spec for sure), ship complete order and not partial orders and/or send on dry ice. For other options, a special handling request can be requested through the Invitrogen Account Manager. Re-suspended primers also ship at Room Temperature (they are stable this way), unless instructed otherwise in the “special handling”.
Note: The primers from Frederick are shipped held in a cardboard holder called a “Fluted Partition”. This is made by a company called Fluted Partition (203-368-2548). The tube primers from Illumina are sent in a tube holder that holds 6 tubes.
HRP Oligo shipping: The storage conditions are 4oC in HRP buffer. We ship on ice packs. The box should be marked with a sticker to refrigerate upon arrival.
STORAGE CONDITIONS
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Recommended storage for lyophilized primers and reconstituted primers is -20°C.
AP Conjugate is shipped in AP storage buffer and should be stored at 4°C.
HRP Conjugate is shipped in HRP liquid storage buffer on ice packs and should be stored at 4°C.
HEX, TET and FAM are very sensitive to light. Store in the dark or use amber tubes. Storage in a black box is recommended. HEX and TET labels are both stable under 30 cycles of standard PCR conditions; however under harsher conditions (high pH, high temp) TET is more stable than HEX.
Dye half life (approximately) when exposed to light
HEX 4.5 hours
TET 2.25 hours
FAM 1.125 hours
For making adapters with 5’modified oligos:
We have limited data to show that in high salt solution at 55°C HRP is stable for 1 hour.
We have no stability data of HRP at 65°C. It has been stated that HRP is less stable than AP
STABILITY
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Lyophilized primers are stable for one year, and will be stable for several months at 4°C. Reconstituted primers at -20°C are stable for at least six months, and they will be stable for a few weeks at 4°C.
AP Conjugate - shipped in AP storage buffer, store at 4°C, stable for at least 12 months.
HRP Conjugate - shipped in HRP storage buffer, store at 4°C, stable for at least 12 months.
QC SPECIFICATIONS
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Most of the modified oligos arel be made at Frederick (see General Custom Primer note for details).
For 25, 50 and 200 nM desalted and cartridge-purified DNA oligos, there is 100% OD 260 analysis. Random samples of 25% of the oligos produced are tested by either Capillary Electrophoresis (all oligos at Illumina and any oligo longer than 45-mers at Frederick) or Mass Spectrometry (Illumina does not do Mass Spec anymore but Frederick still uses it for oligos smaller than 45-mers). DNA oligos that are desalted and ordered at 25 and 50 nM scales also have 100% real-time digital trityl monitoring during analysis. Customers can request 100% QC by Mass Spec or CE during ordering paying $1 per oligo.
Desalted DNA oligos ordered at 1 and 10 uM, DNA oligos at any scale that are purified by HPLC and PAGE, the majority of the DNA oligos with 3 and 5’ modifications and the RNA oligos have also 100% OD260 analysis and Mass Spectrometry or Capillary Electrophoresis.
For a PAGE oligo, regardless of length, the main peak on the trace needs to be at least 85% of the product to pass and the N- peaks for mutations must be less than 10% (Jaime 11.30.06, from Sheryl Moles).
Effect of modification on OD
Oligos are quantified by measuring the absorbance at 260 nm. Some fluorophores do absorb at 260 nm as well. For example, fluorescein and TAMRA have an extinction coefficients of 20.96 and 31.98 OD/μmole at 260 nm. For reference, dA, dC, dG and dT are 15.3, 7.4, 11.8, 9.3 OD/ μmole respectively. So, TAMRA is equivalent to about 3 nucleotides. For the dyes that have a published values at 260 nm, the value is incorporated into the extinction coefficient calculation on the C of A. These modifications are: FAM, Fluorescein, HEX, TAMRA, and TET. Others may absorb at 260 nm, but the data is not published and therefore is not included in the calculation.
OD Specifications for AP and HRP Conjugates- Not applicable because pass/ fail based on moles of conjugate primers. The protein and oligo make up the OD value and may not reflect the true OD value of the oligo.
AP and HRP Conjugates
Bases Scale Desalted Cartridge*HPLC PAGE
<20 Bases25 nmol NA NA NA NA
50 nmol NA NA NA NA
200 nmol NA NA NA NA
1 μmol NA NA1-5 nmole (0.5-13
OD)1-5 nmole (0.5-13 OD)
10 μmol NA NA Inquire Inquire 20 Bases25 nmol NA NA NA NA
or greater50 nmol NA NA NA NA
200 nmol NA NA NA NA
1 μmol NA NA1-5 nmole (0.5-13
OD)1-5 nmole (0.5-13 OD)
10 μmol NA NA Inquire Inquire
*Per Jeremiah Mitchell (03/18/2009) we can't offer any mod on a cartridge purified oligo. The modification interferes in some way with the purification mechanism.
QC on Taqman probes:
Taqman and other Fluorescent probes will be QC’d by Mass Spectrometry (Mass Spec, MS). “They will be run on mass spec. We try to hit the guaranteed amount but we do not make a guarantee.” – Jeremiah Mitchell (04/02/2009)
PROTOCOL AND APPLICATION NOTES
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Modification/scale/purification
Manufacturing Details
Fluorescein
Rhodamine
HEX/TET/FAM
Phosphate
Biotin
Amine
Gateway
Alkaline Phosphatase
Horse Radish Peroxidase
Phosphorothioate
“Fully-Phosphorothioated" Custom Primers
Aldehyde
Acridine
Thiol
Delivery Schedule
OligoPerfect Designed Primers
“Fully-Phosphorothioated" Custom Primers
3' Modifications of Oligos
Reconstitution Protocol
A260/A280 ratio of the oligo
Oligo Visualization
Troubleshooting
“Custom Custom” modifications
Modification, Scale of Synthesis/Purification and Availability Grid (back to Table of Content)
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Mod Synth
Scale Purification
Desalted
Purification
Cartridge
Purification
HPLC
Purification
Gel Purified
5' Biotin25 N Yes (10-50 bases)NO NO NO
5' Phosphate50 N Yes (10-50 bases)NO NO NO
5' Primary200 N Yes (10-50 bases)NO Yes (7 – 60 bases)Yes (7-100 bases) Amine 1 U Yes (10-50 bases)NO Yes (7 – 60 bases)Yes (7-100 bases)
25 U Yes (10-50 bases)NO Yes (7 – 60 bases)Yes (7-100 bases)
25 N NO NO NO NO
50 N Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases) 5' Rhodamine200 N Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases)
1 U Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases)
25 U Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases)
25 N Yes (10-50 bases)NO NO NO
5' HEX/ TET/FAM50 N Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases) 5' Fluorescein200 N NO NO Yes (7 – 60 bases)Yes (7-100 bases) 5' GATEWAY 1 U Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases)
25 U Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases)
25 N NO NO NO NO
AP Conjugate50 N NO NO NO NO
HRP Conjugate**200 N NO NO NO NO
1 U NO NO Yes (7 – 60 bases)Yes (7-100 bases)
25 U NO NO Yes (7 – 60 bases)Yes (7-100 bases)
25 N NO NO NO NO
50 N Yes (5-100 bases)Yes 7 – 60 bases)YES (7 – 60 bases)YES (7-100 bases) Phosphorothioates200 N Yes (5-100 bases)Yes (7 – 60 bases)YES (7 – 60 bases)YES (7-100 bases)
1 U Yes (5-100 bases)Yes (7 – 60 bases)YES (7 – 60 bases)YES (7-100 bases)
25 U Yes NO YES (7 – 60 bases)YES (7-100 bases)
Reason for lower limit of 10 mer on the 25 nmol scale: "We have had a high failure rate on synthesis of short oligos at the 25 nmoles scale. We have determined that the problem cannot be corrected. Therefore, we have decided to make the minimum length at the 25 nmole scale to be a 10 mer.
The amount of modified oligo specified in the CoA includes the modification. For example, for the biotin modification, the amount of oligo in ‘ug’ and ‘uM’ specified in the CoA includes the oligo, the C6 linker and the biotin.
**HRP max oligo length of 25 bases, requires 1 umole scale.
Manufacturing Details
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These modifications are added as phosphoramidites on the machine during synthesis.
Fluorescein
Rhodamine
HEX/TET/FAM
Phosphate
Biotin
Amine
Gateway
Alkaline Phosphatase
Horse Radish Peroxidase
Phosphorothioate
Aldehyde
Acridine
Thiol
5’ FLO Modification (Formerly referred to as Fluorescein)
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5(6)-FAM (5(6)-carboxyfluorescein) is added as a phosphoramidite by B-cyanoethyl chemistry and therefore added on the sugar at the 5' end of the primer NOT on the last base as the last synthesis cycle. This is a mixture of two isomers of FAM (therefore on an HPLC trace, there will be two peaks).
Absorption Max (nm)Emission Max (nm)Extinction Coeff.
(OD/mole) at max
Extinction Coeff. (OD/mole) at
260 nm
49452076,00031,500 Fluorescent color is green.
Note: 5’ FLO is a mixture of the 5- and 6-isomers of FAM. They are not stereoisomers. The conjugation bond is either on the 5 and 6 position on the phenyl ring. Half the molecules have the bond at the 5 position and half have it at the 6 position**. The two different oligos can be detected when analyzed by HPLC or CE. The extinction coefficients at lambda max and at 260 nm are estimates based on known values of the two isomers. This modification is not available desalted or cartridge purified per Sheryl Moles 3/29/07 (V.Gurtu).
The phosphoramidite used for this does not have a trityl group, but the FAM itself is hydrophobic enough to allow HPLC purification. A modified HPLC protocol is used for the 5' phosphate oligos to achieve the separation. Typically, 5' fluorescein oligos will not be as pure as those that can be purified by using the trityl on approach, but close in purity.
FAM and Fluorescein are different chemicals. FAM is 5-carboxyfluorescein or 6-carboxyfluorescein (single isomers) or 5(6)-carboxyfluorescein (mixed isomers) – FAM has two carboxyl moieties on the phenyl ring, while Fluorescein has only one carboxyl on the phenyl ring; all reactive groups will be conjugated via the 5- or 6- carboxyl group on FAM but directly to the phenyl ring on Fluorescein. Although these two dyes have very similar spectral properties, the names “FAM” and “Fluorescein” are not interchangeable. FAM, not Fluorescein, is used on ABI sequencers. The two isomer forms of FAM differ in electrophoretic mobility and both FAM isomers differ significantly from the electrophoretic mobility of Fluorescein.
Specs for FLO/FAM mods:
“We will use 5'FLO amidite (6-FAM)so there is no free dye.
If the oligo is desalted it has to have at least 45% full length product (note that these specs are not public knowledge) and contain no N- peaks that are greater than 15%.” – Jeremiah Mitchell (05/19/2009)
*[Note: Highly concentrated solutions and the solid forms of fluorescein and FAM appear by eye as yellow or orange colored solutions or solid, this may also be true of lyophilized oligos labeled with 5’FLO].
** “Half the molecules have the bond at the 5 position and half have it at the 6 position”. Our mixed isomer FAM products, 5(6)-FAM, can have a lot-to-lot variablility in 5:6 isomer ration from 70:30 to 50:50; is the 5(6)-FAM phosphoramidite SPECIFICALLY provided always a 50:50 ratio of isomers, or is there lot-to-lot variability as well? It is possible, so you can not always tell customers it is 50:50. (O. Cholewa, 8/2008).
This modification can be added to phosphorothioated oligos.
Rhodamine
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Can suggest as the alternative to 5’ROX modified oligos.
All the rhodamine dyes (rhodamine, rhodamine green dye and rhodamine green-X) are HPLC purified as part of the dye price. The web site forces you to order it as desalted for now (8.4.06) but it will be changed in the future.
Rhodamine when resuspended in water or TE solution will appear pink-red-purplish.
Rhodamine = RHD on email order form.
MW(NH4 salt)MW (no salt)Emission
Abs. (nm)Extinct. Coeff. (at lambda max)**
Max (nm)
743.8726.859657292,000
Note that this is for measurements of the labeling dye in methanol. The values in water/TE and attached to an oligo will be different.
** We do not have info on what A260 of rhodamine would be.
The modification provided is x-rhodamine isothiocyanate (X-RITC) which is a mix of rhodamine-5 and 6-isothiocyanate (5(6))-XRITC). The addition of the rhodamine modification is done on all scales as a post-synthesis modification. To see the structure, click on the link in this paragraph.
HEX, TET or 6-FAM
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HEX is 4,7,2`4`5`7` hexachlorofluorescein
TET is 2`7`4,7, tetrachlorofluorescein
FAM is 6-carboxyfluorescein, while fluorescein is the 5 and 6 isomers mixed together.
FAM, HEX and TET are added as a phosphoramidite as the last synthesis cycle using B-cyanoethyl chemistry and therefore added on the sugar at the 5'end of the primer NOT on the last base. They are covalently attached to the 5' end of the last sugar via a phosphodiester bond.
Point at which modification occurs:
In synthesis we can add HEX, TET, FAM or flourescein. All other dyes are added post-synthesis. In synthesis mods do not require purification. (Jeremiah Mitchell, Melissa Almeida; 04/30/2009).
Trityl-OFF cartridge purification on confirmation sheet refers to the fact that there is no DMT on the phosphoramidites with the dye.
For Eurogentec orders take the MW from the CofA and add the below MW numbers. PrimerTrak includes the MW of the Mod in the MW from the CofA.
MW Emission Max
(nm)Excitation (Abs)
Max (nm)
Extinction (at max)Extinction (at 260)
FAM554.551749474,85020,960
TET692.253852285,55316,255
HEX761.155353595,69831,580
Molar extinction coefficients above are measured at excitation maximum nm.
Individual experiments may vary fluorescence intensity due to minor variations in pH or composition can affect the above numbers. For FAM, take the reading at pH 9 since at this pH the absorbance is highest. At pH 7, this value is reduced by 16% for FAM. And when the fluorophore is attached to DNA, the value is also reduced.
We do not know the effect of pH on the absorbance of TET & HEX.
At the maximum wavelength, there is a huge change between pH 8 and pH 6.5. For the absorbance at 260, the change between pH
8 and pH 6.5 is relatively minor.
I tested the water here and it is about 6.5.
Also please see https://www.doczj.com/doc/0c17643869.html,/handbook/figs/fig23-2.html
for more information on effect of pH.
The color of the dye depends on the "Filter-wheel set" of the ABI instruments:
Dyes Filter A Filter B
HEX Green (560nm)Yellow (560nm)
6-FAM Blue (531nm)Blue (531nm)
TET-Green (545)
Color in solution: HEX is pinkish, FAM the yellow, and TET is orange, but if the oligos are not subjected to light they may appear colorless. 10nm often will have no color due to the small amount of oligo present.
The phosphoramidites with these dyes do not have a DMT (trityl) group. They therefore cannot be monitored during the synthesis with trityl analysis. They still must be deprotected, as there are protective groups on the bases in the oligonucleotide. A & C have a benzoyl protective group. G has an isobutyl protective group. T does not have a protective group.
Trityl-OFF cartridge purification is required since these do not have a DMT. This purification is performed as a batch HPLC method on a polystyrene reverse phase support.
They cannot be ordered as phosphorothioates.
Phosphate
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5’- Phosphate
The cartridge purification option for the 5' phosphate oligos is not offered, because it is not technically possible (cartridge purification relies on the DMT group being present which is not true here). DMT has molecular weight of 79.98.
The 5' phosphorylation is added by B-cyanoethyl chemistry and therefore added on the sugar at the 5'end of the primer NOT on the last base, as the last cycle with a phosphoramidite. It therefore has a trityl group, which is released and monitored to assess the coupling efficiency. (During the coupling, the phosphate has a protecting group, which is removed prior to cleavage of the oligo from the solid support). Since 5' phosphorylation comprises the final step after all the cycles are complete, only the complete, or full length oligos (which are uncapped) can be phosphorylated on the synthesizer.
Gel purification of phosphorylated primers will not purify full length primers from n-1 primers because the phosphate adds an additional negative charge that affects electrophoresis.
The 5’ phosphate oligos are purified by HPLC by size. A modified HPLC protocol is used for the 5' phosphate oligos to achieve the separation. Typically, 5' phosphate oligos will not be as pure as those that can be purified by using the trityl on approach. The cartridge purification option cannot be used for the 5' phosphate oligos, as this relies on the DMT group being present.
In fact, when the yield of HPLC phosphorylated primers is low it probably was because there were a lot of failure sequences so not as many fractions could be combined. There would be a lot of multiple overlapping peaks in preps with more "n-x" failures. During HPLC purification, 5 Phosphate oligos are not DMT-selected.
It’s been noticed that a couple of phosphate, HPLC oligos after speedvac are a bit "dirty", or slightly yellow. This is sometimes a product of the synthesized product. It is usually cleaned up in the ethanol precipitation step as part of QC.
3’-Phosphate
The 3’ phosphate is added attached to the solid support so the sequence is actually coupled to it in the first round of base addition. It will block extension by polymerases. Stability: assuming it's DNA, 3'-PO4 is more stable than 3'-BIO. 3'-BIO will come off under basic condition while 3'-PO4 will not.
Biotin
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The structure as shown is called Biotin Phosphoramidite. Biotin is on the far right ending with the oxygen group next to the amine group in the chain. MW of biotin is 405. Biotin oligos are quantified as normal by OD260 (biotin does not absorb at 260). Comparing 3’ Mod Stability: 3'-NH2 is slightly more stable than 3'-PO4. It is 3'-NH2>3'-PO4>>3'-BIO
The biotin that we use for our modifications is from Glen Research, 10-5950-95 (Jeremiah Mitchell, 04/27/2009).
5’- Biotin
PROPRIETARY INFORMATION: It is purchased from Glen Research and Catalog number 10-5950-02.
https://www.doczj.com/doc/0c17643869.html,/ Please see the biotin structure below.
Biotin dT The Biotin dT has the Biotin is attached to a dT base, so they would only use it if the 5' base can be T. I really know no reason why one would choose it over the regular biotin.
General info on Biotinylated primers:
5' biotinylated primers may work for chemiluminescent detection, but TdT tailing would be better.
The biotin is added during synthesis, before the purification step. A biotinylated oligo (or a PCR product made with biotinylated oligos) will be retained for a longer time on a reverse phase HPLC column than an unbiotinylated oligo of the same size. This is due to the hydrophobicity of the biotin moiety.
The biotin modification should be fairly resistant to heat and pH (as it has been boiled to 100°C and exposed to pH 2-10). More extreme conditions may be tolerated, but have not been tested. Presence of reducing agents may not be a problem, but exact limits are unknown.
The chemical structure of 3'-Biotin predicts that 3'-Biotin would come off/degrade at two conditions: 1 over the time, 2 the oligo was exposed to basic conditions such as dissolved in basic buffer. The reason is that 3'-Biotin has an adjacent hydroxy group which makes it a RNA-like structure.
5'-Biotin though does not have this adjacent hydroxy group, so it is pretty stable, more stable than 3'-Biotin.
The standard biotin that we use has a C6 linker. We have used the TEG biotin linker several times in the past because of special requests from customers but it is not our “standard” biotin modification (Jaime 5/10/06 from Joseph Hayes).
Recover 5’ biotin labeled oligo from C-18 column: An oligo with biotin is more hydrophobic than an unlabeled oligo. Therefore, the biotin oligo would stick better to a C-18 column. I haven’t used a C-18 column for years since the polystyrene columns are more durable for production. To put it in perspective, on the polystyrene I elute the unlabeled oligos in 6 - 12 percent
ACN/TEAA. For the biotin oligos I use 10 - 15 %. The customer may be able to use this to extrapolate the percentages. Since C-18 is more hydrophobic than polystyrene, their percentages should be a little higher.
Frederick QC’s the oligos by Mass Spec or CE and they check the peaks to see if the coupling reaction of the biotin to the molecule went as expected. For a DSL oligo, we will discard the oligo if there is a peak indicating more than 15% of the oligo not coupled to biotin. For HPLC or PAGE purified oligos, we will ask for less than 10% of uncoupled oligo during QC (Jaime from Sheryl Moles, 12.14.06).
Biotinylated DNA will successfully transform bacteria, e.g. DH5 alpha.
3’- Biotin
3’ Biotin is added via biotin solid support.
Primers carrying a 3’-biotin modification can not be longer than 99 nucleotides. Since the 3’-biotin is provided as a CPG modification (linked onto the solid support) the system interprets the 3’-biotin as the last nucleotide of the sequence. (Sheryl Moles 02/08/07)
Comparing 3’ Mod Stability: 3'-NH2 is slightly more stable than 3'-PO4. It is 3'-NH2>3'-PO4>>3'-BIO
Glen Research Catalog Number: 20-2955-xx https://www.doczj.com/doc/0c17643869.html,/
Primary Amine
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Primary amine –The amino group is linked to the 5' end via a 6 carbon spacer arm. i.e. Off of the 5’ hydroxyl there is a phosphate. Attached to this via a phosphodiester bond is a C6 linker (CH2-CH2-CH2-CH2-CH2-CH2) followed by a primary amine NH2. It is added as a phosphoramidite at the last synthesis cycle by B -cyanoethyl chemistry and therefore added on the sugar at the 5'end of the primer NOT on the last base.
For other scales, we can also do 3’ amine modification as a custom “custom” through PMO request (Jeremiah Mitchell). 3’ Amine mod has a C7 linker. We can now do 3’ amine modification as a catalog item for 50 nmol and 200 nmol scale. Customers will be able to order online, or using the email/fax form in North America. The website will be updated with the proper drop-downs for the mod, and the forms will be updated with the code. Please note that the 3’ amine mod is only compatible with certain 5’
modifications, ie, FAM, HEX, TET, Fluorescein, Biotin, Amine, and phosphate. The other 5’ mods (such as the Alexa dyes) require amine to add them to the oligo and so there would not be any way to prevent the dye from also attaching to the 3’ end. Pricing for the 3' mod AMINE for 50 and 200N:list price $14 and $30 USD respectively.
Comparing 3’ Mod Stability: 3'-NH2 is slightly more stable than 3'-PO4. It is 3'-NH2>3'-PO4>>3'-BIO
(When primary amine-linked oligos are made, an amino link is used with a TFA (trifluoroacetyl) protecting group. Using the TFA aminomodifier does not give any method of detecting the coupling, i.e. no trityl. The only method is CE. This will tell what percentage of label is on the oligo, but if it is near 100% without label, it will look like 100% labeled. The linker is the same as the MMT version (6 Carbon linker). All other comments on the amino oligos are the same as the MMT manufactured type since the products are ultimately identical. )
The 5’ AMN fro ILMN is C6 amine linker (5’ MMT). The linker from Frederick is TFA.
C6 amine dT (from the Resgen offering) The 3 letter code for amine C6 dT is AM6.
Typical coupling efficiency for the addition of the 5' amine is >95%, and the amine group will be transparent at 260 nm; it absorbs at ~210 nm. Its presence really cannot be monitored by electrophoresis (on agarose or acrylamide).
If custom oligos is to be attached to glass slides for the purpose of microarrays, TeleChem International Inc. (https://www.doczj.com/doc/0c17643869.html,) may supply the chemicals for DIY, or the custom oligos (made by Invitrogen) can be modified with a 5' mod C6 amine and stuck to glass slides for microarrays. We can refer our custom primers customers to this site for immediate microarray needs. An "arraying buffer" needs to be obtained from this company. The custom oligonucleotides should be resuspended in this buffer at a specified concentration, and supply 3 μl of this resuspended oligo in a 384-well plate to the company. TeleChem can fix the oligos to the slide as a service.
To verify the presence of the 5’ amine ordered, please see the excerpt from the Clontech manual for N-TFA-C6-Aminomodifier Below.
Measurement of Primary Aliphatic Amines
Two convenient protocols are recommend that described in the literature that are useful in measuring aliphatic primary amine group (Smith, L.M., et al. (1985) Nucl. Acids Res. 13:2399; Weigele, M., et al. (1972) J. Amer. Chem. Soc. 94:5927; Weigele, M., et al. (1972) J. Amer. Chem. Soc. 94:5927).
Smith et al. (3) quantitatively measured the presence of the 5' aliphatic primary amine-modified oligonucleotide by reacting with fluorescamine and employing a fluorescent excitation wavelength of 390 nm and emission wavelength of 487 nm (Weigele, M., et al. (1972) J. Amer. Chem. Soc. 94:5927).
Alternatively, aliphatic primary amines may be quantitatively measured using UV absorption at 570 nm by a ninhydrin test (Weigele, M., et al. (1972) J. Amer. Chem. Soc. 94:5927). For qualitative work, the ninhydrin test is probably the most convenient. When measuring aliphatic primary amine groups on aminomodified oligonucleotides, it is important to use a sample uncontaminated by other amines. Interfering ammonium ions present from the deprotection steps can be removed by treatment of the aminomodified oligonucleotide with Dowex AG50W-X4 (cation exchange resin, sodium form) for 30 minutes at room temperature (Weigele, M., et al. (1972) J. Amer. Chem. Soc. 94:5927).
Qualitative Test for Primary Amine Groups
Spot 0.15 OD (6 ug) of aminomodified oligonucleotide on a silica gel TLC plate. Next to it, spot an equivalent amount of an unmodified oligonucleotide to be used as a negative control.
Carefully dry plate with heat gun or hot plate. Cool.
Spray with a ninhydrin solution (0.2% ninhydrin, 2% sulfuric acid in ethanol).
Dry and carefully heat plate with a heat gun or hot plate. Be careful not to overheat. A positive will result in a blue or purple spot. To block ligation or polymerase extension, it is normally thought to use a dideoxy terminator, which is costly or not readily available. Since all that is needed is not to have the 3' or 5', depending on application, adding a 3' or 5' amine modification will accomplish the task. Additionally, amine modifications are the simplest and least expensive of the modification reagents. Any of
the amine modifications will work. A C6 version for the 5' end, works fine. 3' amine modification is not available through Invitrogen.
To crosslink double-stranded DNA with a peptide using chemical crosslinkers: A 5' modification with a primary amine and cross link with the peptide with glutaraldehyde can be tried.
Gateway attB1, Gateway attB2
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These are essentially general custom primers, but have a set grouping of nucleotides at the 5' end. For technical suggestions on designing Gateway primers, refer to the Gateway Technology handbook.
attB1 oligo sequence: GGGG ACA AGT TTG TAC AAA AAA GCA GGC T (29 bases)
attB2 oligo sequence: GGGG AC CAC TTT GTA CAA GAA AGC TGG GT (29 bases)
Alexa
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Alexa modifications required to be HPLC purified. The available Mods are 488, 532, 546, 555, 594, 647, 660, 750. They are all attached to the oligo using a C6 linker. These are also available at 50 nmole and 200nmole only at this time. Please contact Jeremiah Mitchell for larger scales.
Link to Alexa Dye info on our website:
https://www.doczj.com/doc/0c17643869.html,/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/alexa-fluor/Alexa-Fluor-Dyes-Across-the-Spectrum.html
Q&A for Alexa Dyes:
1. What is the minimal length of Alexa-labeled oligo that we can synthesize? (He wants to label short oligos, 6-mer and longer). Through our standard manufacturing group the shortest length we offer is a 10mer. We would be willing to try to make an alexa labeled, 6mer through our custom services group.
2. He wants to know if the fluor dye can be put on at the 3' end of the oligo?
Yes we will attach the alexa to the 3' prime end using an amino.
3. He asked if the HPLC purification is mandated for synthesizing Alexa fluor labeled oligo?
Yes
4. He asked if the Alexa fluor dye is added during the synthesis of the oligo or after the synthesis of the oligo
The alexa dye is added post synthesis.
Other dyes
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Bodipy dyes, Marina Blue, Oregon Green dyes, Pacific Blue, Rhodol green and Texas Red-X are modifications that required purification HPLC at least to remove the excess of dye.
How is the modification added? First, we add an amino group to the phosphate at the 5' end of the DNA, then the amino is linked to one of Bodipy's carboxyl group.
The Oregon Green 500 dye modification is no longer available (Nov 2008), although the web may not be updated with this information yet. Texas Red is no longer available (April 2010, not reflected in website yet.)
Alkaline Phosphatase
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Offered as through standard ordering, requires 1 μmole synthesis and HPLC purification (only) (Jeremiah Mitchell – 10/23/2009)
AP Conjugates are shipped in liquid storage buffer (3M NaCl, 1 mM MgCl2, 0.1 mM ZnCl, 0.02% NaN3, 30 mM triethylamine, pH 7.8). They are clear in liquid, and HPLC or PAGE purification is required in order to remove any unreacted dye material. The alkaline phosphatase used is Calf Intestinal Alkaline Phosphatase.
The 5'-AP-oligonucleotides are synthesized with a 5'-end-thiol hexyl linker. The thiol group is the site of attachment between the AP and oligonucleotide. This modification also functions as the "spacer arm" between the oligonucleotide and the enzyme. Activated AP is then reacted with the 5'-end thiol group, and the conjugated oligonucleotide is purified by ion-exchange HPLC to remove the un-reacted products. To verify the conjugate meets specifications, its activity is measured and final yield is quantified by UV.
Substrate for ELISAs :
P-nitrophenylphosphate(pNPP) and the emission wavelength for monitoring reactions is 405 nm.
Substrate for Immunoblotting and Immunocytochemistry:
5-bromo-4-chloro-3-indoylyphosphate p-toluidine salt (BCIP) and nitroblue tetrazolium chloride (NBT) and the color of the precipitated product is blue/purple.
naphthol-AS-phosphate and the color of the precipitated product is reddish purple.
naphthol-AS-BI-phosphate and the color of the precipitated product is reddish purple.
naphthol-AS-BI-phosphate and fast red TR and the color of the precipitated product is red.
naphthol-AS-BI-phosphate and new fuchsin and the color of the precipitated product is crimson red.
pyronin Y and the color of the precipitated product is reddish purple.
Horse Radish Peroxidase
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Horseradish Peroxidase conjugated oligo is shipped in liquid storage buffer (1M NaCl, 0.1M NaP pH 7.3, 0.01% thimerosal). The specific activity will be in the range of 15,000 - 25,000 U/mg. The oligo will have a tan/brown tint in liquid, and HPLC or PAGE purification is required in order to remove any unreacted dye material.
Pricing is not on the web. We only do HPLC 1U HRP and the price for the mod is $125 in USA and $160 in Canada as of 2008.
These are made by Eurogentec. They can’t really do HRP oligos over 25bp but our system still accepts those orders, which may lead to problems when the oligos fail. We are considering discontinuing the modification or bringing it in house if possible. Substrate for ELISAs:
5-aminosalicylic acid (5AS) and the emission wavelength for monitoring reactions is 450 nm.
2,2 azino-di-(3-ethylbenzthiazonine sulfonate) (ABTS) and the emission wavelength for monitoring reactions is 410 nm.
o-dianisidine and the emission wavelength for monitoring reactions is 403 nm.
o-phenylenediaminedihydrochloride (OPD) and the emission wavelength for monitoring reactions is 492 nm.
3,3,5,5-tetramethylbenzidine (TMB) and the emission wavelength for monitoring reactions is 450 nm.
Substrate for Immunoblotting and Immunocytochemistry:
3-amino-9-ethylcarbazone (AEC) and the color of the precipitated product is reddish brown.
4-chloro-1-napthol (4CN) and the color of the precipitated product is blue.
3,3-diaminobenzidine tetrahydrochloridedihydrate (DAB) and the color of the precipitated product is brown.
DAB with nickel enhancement and the color of the precipitated product is blue/black.
Hanker-Yates Reagent and the color of the precipitated product is blue/black.
naphthol and the color of the precipitated product is pinkish red.
3,3,5,5-tetramethylbenzidine (TMB) and the color of the precipitated product is greenish brown.
Phosphorothioate (S-oligos)
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S-oligos (sometimes referred to as phosphorothioates or S-ODNs) are oligonucleotides with a sulfur substituted for one of the oxygens in the phosphodiester bonds between the nucleotides. The modification is done at the linkage level (as opposed to post synthesis). One base's linkage to all of the phosphodiester bonds in the oligo can be ordered sulfur derivatized. The 3' base cannot be ordered as a phosphorothioate because the 3' position is always an OH once it has been cleaved off the solid support. An HPLC trace of a purified phosphorothioate oligo may look like a doublet or a very broad band. The base is added and then the linkage is modified. The phosphate between the two bases is converted to a double bond "S" (using Beaucage reagent) instead of the regular double bond "O" (using an iodine solution). These oligos are not synthesized in a stereo specific manner, the final product is a mixture of the R and S stereoisomers about the sulfur bearing phosphorus atom (Sheryl Moles, 05/24)
We DO offer 5' mods to S-oligos. i.e a customer can order a fluorescein labeled S-oligo. Contact Sheryl Moles for what modifications are available. Also we cannot offer phosphorothioates on any other portion of an oligo while leaving the other bases “normal.” This is due to legal issues and not chemistry. Customers desiring phosphorothioated nucleotides at the 5’ and 3’ regions of a primer and no modifications to the nucleotides in the middle must order their primers from Integrated DNA Technologies, Inc.
Use of S Oligonucleotides for Antisense Applications:
This entry provides an overview for the use of S-Oligonucleotides (S-ODNs) for antisense work. Faxable copy in
l:\oligos\antisense delivery overview.doc
Antisense phosphorothioates can function in vivo by a number of different mechanisms. They can work at the RNA level to inhibit reverse transcriptase activity (to inhibit RNA virus replication) or by generating a substrate for RNase H activity [S-ODNs form a duplex with RNA which still renders the RNA accessible to RNase H cleavage]. Antisense oligonucleotides can form triple helices, which in turn interferes directly with transcription or can also inhibit translation. S-Oligos tend to be less efficient substrates for nucleases present intracellularly and in serum/media. [Akhtar(1991)Life Sciences 49, 1793]. Because S-ODNs maintain the same charge state as underivatized oligonucleotides, they are capable of complexing with cationic lipids for delivery into cells. They therefore also suffer from poor passive permeability to cells in culture media.
Overview of antisense technology:
Antisense oligos are used for the selective regulation of gene expression in living cells (or in invitro translation experiments). This technique is based on blocking the flow from DNA to protein via RNA by introducing a complementary sequence to a portion of the target RNA. Generally the position of the antisense oligo in the target mRNA can alter the effectiveness of the antisense oligo (e.g. oligos complementary to the translation initiation codon region of mRNA tend to work well).
In general oligonucleotides should meet the following criteria to be effective as antisense primers:
They should be chemically stable
They should be very stable in aqueous solutions
The RNA/DNA hybrid should have the same (if not better) thermostability (Tm) as unmodified oligos.
They should be nuclease resistant.
Next to phosphorothioates, methylphosphonates (O Methyl Oligos) tend to be the next most widely used oligonucleotide derivative for antisense work. A disadvantage of methylphosphonates is that they are NOT substrates for RNAse H and therefore are not as effective in catalyzing the degradation of target mRNA. Additionally methylphosphonates exhibit decreased solubility in aqueous solutions and also lose their negative charge because the oxygen on the phosphorous is substituted with a methyl group.
Generalizations/Design Suggestions for S-ODNS:
Typical Size Range: 14-30 mers. (Most publications used S-ODNs as 20 mers. This corresponds to a mass range of 5000-11000.
Concentration Range (in Culture Medium): 15-25 μM (Concentrations greater than this can cause nonspecific toxicity with S-ODN's.)
Concentration Range during Lipid-Mediated Delivery: 0.1-5 μM. (The optimum concentration of oligo needs to be determined for each cell line AND each molecular target.)
Amount of S-ODN to Use in Microinjection: 0.03-1 μg/μl
Amount of S-ODN for in vivo Injection: 0.05-1 mg S-ODN/Kg of animal.
Number of Phosphorothioate Bonds/Oligo: Generally most references use phosphorothioate linkages between every base.
Recommended Purity: Desalted or HPLC or Multiple HPLC (reverse phase and anion exchange HPLC).
[Most references actually use HPLC purified primers for antisense work. One reference (Ic.) actually compared HPLC and desalted oligos and found no difference). Additionally, however some references reported the sterilization of 10 uM stocks of oligos by centrifuging thru 0.2 um Centrex cellulose acetate filters, available from Schleicher & Schuell].
Multiple HPLC is not offered.
Potential Antisense Primer Design and Sequence Selection Strategy:
Choosing an effective sequence to use for antisense work cannot necessarily be done by looking at the results from an oligonucleotide selection program. Even knowing the secondary structure for the oligonucleotide will not necessarily mean it will be effective at affecting gene expression. Typically, several overlapping oligos are made within a gene sequence and each one is tested for binding to the target mRNA [by making a chip-based oligo array and hybridizing radioactive RNA to it or by Northern blot analysis (using the labeled oligo as a probe). Only about 1 in 10 show good binding in high stringency conditions. At this point, hybrid oligos are often used that combines the benefits of the different oligo chemistries. 2'-O-Methyl derived nucleotides (less toxic and high affinity) is used to synthesize the 3' and 5'ends of the primer and phosphothioated nucleotides (more toxic and lower affinity, but excellent substrates for intracellular RNase H) are used to make up the middle portion of the primer.
You can order 2'-O-Methyl derived nucleotides in your oligo, but at the current time this can only be done through the email spreadsheet and not online. To specify such a modified base, enter the base in brackets like this [MeG][MeC][MeU][MeA]
….then the rest of your sequence as normal. However they can only be order for RNA and I don't think that this is listed on the web anywhere. If an external customer needs 2-o-methyls they should be ordered through 999Q. If an internal customer needs 2’-O-methyls they can use the email form but they need to be aware that there is a 150 character limit in E1. This means that the longest 2’-O-methyl RNA that can be synthesized is a 30mer. Since [MeA] has five characters. (J. Mitchell 4-2010)
DNA/RNA hybrids have to come through 999Q. We can use 2’O- methyls or standard RNA for the hybrid. (J. Mitchell 4-2010)
Improved transport through the cellular membrane can be achieved by use of a carrier molecule or by backbone modification to more lipophilic linkages (methylphosphonate). Increasing the affinity and specificity of an oligonucleotide has been more difficult to achieve, since this necessitates modifying the natural bases, which are already set up almost perfectly for optimal hydrogen bonding. C5-propyne derivatives are at the moment the best commercially available alternatives.
We do not sell these C-5 propyne oligos.
Antisense oligodeoxynucleotide phosphorothioates in which all dC and dT residues have been substituted by the C-5 propyne analogues, pdC and pdU, have been shown to be effective inhibitors of gene expression as a result of enhanced binding to the target RNA sequence (Wagner, R.W. et al., Antisense gene inhibition by oligos containing C-5 propyne pyrimidines. (1993) Science 260, 1510-1513). Duplex stability and thus Tm can be raised using modified bases. For each replacement of dT with propyne dU, the duplex melting temperature is increased by 1.7 C. The elimination of water molecules from the duplex is believed to cause improved stacking. Evidence suggests that the strength of the oligo:mRNA hybrid is increased if C-5-propyne modified nucleotides (i.e. 5-(1-propynyl) 2' deoxyuridine or 5-(1-propynyl)- 2'deoxycytidine) are used.
Other references for C-5 propyne analogues:
Flanagan, W.M., L.L. Sun and R.W. Wagner, Elucidation of gene function using C-5 propyne antisense oligonucleotides, Nature Biotechnology, 14, 1139 (1996)
Flanagan, W.M., A. Kothavale and R.W. Wagner, Effects of oligo length, mismatches and mRNA levels on C-5 propyne modified antisense potency, Nucleic Acids Research, 24, 2936 (1996)
Moulds, C. et al. Site & mechanism of antisense inhibition by C-5 propyne oligonucleotides, Biochemistry, 34, 5044-5053 (1995)
Fenster et al., Inhibition of HIV-1 env expression by C-5 propyne oligonucleotides specific for Rev-response element stem-loop V, Biochemistry, 33, 8391-8398 (1994).
Use of Lipids to Deliver Antisense Phosphorothioates
Invitrogen is licensed to support this application.
Please see Lima, W., and Crooke, S. (1997) Biochemistry, vol.36, pp. 390-398. Binding Affinity and Specificity of E. coli. Rnase H1: Impact on the Kinetics of Catalysis of Antisense Oligonucleotide-RNA Hybrids.
Phosphorothioated ODN were shown to serve as a substrate for RNase H when bound to its complementary RNA, while 2 methoxy (OCH3), and other 2 prime modified ODN, do not support RNase H activity when bound to their complementary RNA.
A strategy for anti-sense gene therapy makes use of this by capping one or both ends of the oligo with 2 methoxy modifications while the remaining sequence gap consists of phosphorothioates.
The mechanism of lipid-mediated delivery of fluorescein-labeled phosphorothioate ODN in CV-1 cells (Green African Monkey Kidney Cells) is discussed. Results indicate that ODN nuclear accumulation begins about 30min after addition of the complexes to the cells. By 3hr postincubation, the ODN are localized predominately in the nucleus, whereas the lipids are distributed in the cytoplasm. The authors also looked at the effects of negatively and positively charged molecules and lipid-mediated transfection.
A schematic representation of the ODN/lipid complex uptake pathway and ODN release is presented.
Bennett, C., Chiang, M., Chan, H., Shoemaker, J., and Mirabelli, C.(1992)Molecular Pharmacology 41, 1023. Cationic Lipids Enhance Cellular Uptake and Activity of Phosphorothioate Antisense Oligos.
The use of Lipofectin to deliver S Oligos was found to increase by at least 100 fold the potency of an antisense oligo. Lipids delivered more oligo to the cytoplasm and to the nucleus.
Felgner, J., Bennett, F. and Felgner. P. (1993)Methods: A Companion to Methods in Enzymology 5, 67. Cationic Lipid Mediated Delivery of Polynucleotides.
A protocol is presented for antisense oligonucleotide transfection. Suggested conditions are given for HUVEC and A431 cells with ISIS 1570, an antisense oligo to the AUG initiation codon of the human intercellular adhesion molecule (ICAM).
Chiang, M., Chan, H., Zounes, M., Freier, S., Lima, W. and Bennett, F.(1991)JBC 266, 18162. Antisense Oligonucleotides Inhibit Intercellular Adhesion Molecule 1 Expression by Two Distinct Mechanism.
The affect of position of the antisense oligo in the target mRNA is discussed. This reference compares the use of desalted S-ODNs to trityl-on HPLC purified S-ODNs. No significant differences between the potency of precipitated or HPLC purified oligos were seen!
Yeoman, L., Danels, Y. and Lynch, M. (1992) Antisense Research and Development 2: 51-59. Lipofectin Enhances Cellular Uptake of Antisense DNA While Inhibiting Tumor Cell Growth.
Albrecht, T. et al. (1996) Ann Hematol (1996) 72: 73-79. Transfection with S-oligos (ODN oligonucleotides.) Oligonucleotide concentrations used were 7 to 30 uM. 200,000 cells/well were grown in serum-free medium in the presence of optimal concentrations of SCF and G-CSF for 24 h. 5 ul of LipofectAMINE at 2 mg/ml and different concentrations of ODNs were incubated for 30 min at room temp with 200 ul culture medium. Cells were incubated with the LipofectAMINE oligonucleotide complex after washing in medium for 3 h at 37oC. After washing, cells were cultured for 24 h and further processed for analysis.
Use of Microinjection to Deliver Antisense Phosphorothioates
Dagle, J., Walder, J. and Weeks, D. (1990) Nucleic Acids Research 18, 4751. Targeted Degradation of mRNA in Xenopus oocytes and Embryos Directed by Modified Oligonucleotides: Studies of An2 and Cyclin in Embryogenesis.
Researchers combine modified internucleoside linkages (e.g. methyl-phosphonates) at the 3 and 5 ends with internal phosphorothioate linkages to inhibit translation in early embyrogenesis.
Cazenave, C., Stein, C., Loreau, N., Thuong, N., Neckers, L.Subasinghe, C., Helene, C., Cohen, J. and Toulme, J. (1989) Nucleic Acids Research 17, 4255. Comparative Inhibition of Rabbit Globin mRNA Translation by Modified Antisense Oligodeoxynucleotides.
Woolf, T., Jennings, C., Rebagliati, M. and Melton, D. (1990)Nucleic Acids Research 18, 1763. The Stability, Toxicity and Effectiveness of Unmodified and Phosphorothioate Antisense Oligodeoxynucleotides in Xenopus oocytes and Embryos.
In oocytes, phosphorothioates are more stable than unmodified oligos and are more effective at degrading specific mRNAs.
Bijsterbosch, Martin K., et. al. (1997) Nucleic Acids Research, vol. 25., no.16, pp 3290-3296. In vivo fate of phosphorothioate antisense oligodeoxy-nucletotides: predominant uptake by scavenger receptors on endothelial liver cells. The authors injected S-ODN into the kidney and liver of mice at concentrations of 0.05 - 1 mg S-ODN/Kg of animal. No S-ODN was detected in the urine (the oligo was radiolabeled) when assayed up to 90min. post injection. At higher concentrations (up to 50 mg S-ODN/Kg animal) S-ODN saturate the ability of the target organ cells to take up oligo.
Use of Antisense Phosphorothioates to Inhibit in vitro Translation
Cazenave, C., Stein, C., Loreau, N., Thuong, N., Neckers, L.Subasinghe, C., Helene, C., Cohen, J. and Toulme, J. (1989) Nucleic Acids Research 17, 4255. Comparative Inhibition of Rabbit Globin mRNA Translation by Modified Antisense Oligodeoxynucleotides.
Sequence specific inhibition of translation is reported with S-ODNs (in the nM concentration range), however nonspecific inhibition of protein translation at high [ODN] was reported.
Review Articles
van der Krol, A., Mol, J. and Stuitje, A. (1988)Biotechniques 6.10, 958. Modulation of Eukaryotic Gene Expression by Complementary RNA or DNA Sequences.
A complete review, which describes natural and artificial antisense down regulation of gene expression by antisense oligonucleotides, RNA or genes. Of interest are annotated tables describing the effects on eukaryotic gene expression by phosphorothioates and the resistance/sensitivity of phosphorothioates to nucleases/RNases (respectively).
Akhtar, S., Kole, R. and Juliano, R. (1991)Life Sciences 49, 1793. Stability of Antisense DNA Oligodeoxynucleotide Analogs in Cellular Extracts and Sera.
Stability of phosphorothioate, methylphosphonate and alternating methylphosphonate and phosphodiester internucleoside linkages was studied in HeLa Cell Nuclear Extracts, S100 cytoplasmic extracts, human sera, and calf serum at 37oC was studied. Alt MP, MP and S Oligos were generally of comparable stability and more stable than unmodified oligos.
Brysch, W. and Schlingensiepen, K. (1994)Cellular and Molecular Neurobiology, Vol. 14, No. 5, 557. Design and Application of Antisense Oligonucleotides in Cell Culture, in Vivo, and as Therapeutic Agents.
This reference suggests that toxic synthesis by products are not always adequately removed by trityl on HPLC if the primer will be used at high concentrations. They also suggest that ammonium ions if present from cruder stocks of oligos can inhibit cell proliferation. This reference nicely summarizes the controls that should be designed for antisense experiments.
“Fully-Phosphorothioated" custom oligonucleotide
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The phosphorothioate modification is actually a modification to the normal phosphodiester bond linkage between bases. The codes used to indicate phosphorothiate bases are F, O, E and Z, representing A,C,G, and T respectively. For instance, "E" means a G followed by a downstream phosphorothioate modification linkage which links the G to the downstream (next) base. Therefore, chemically it is not possible to have a "phosphorothioate" as the 3' most base, since there is no base downstream of it. The F, O, E, or Z codes should therefore not be used in the 3' most position when placing an order for S-oligos. Use the normal A, C, G, or T codes in this position.
Aldehyde
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Aldehyde 5’ modification is aromatic. It contains a benzene ring. The MW is 245.
Acridine
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Oligos bearing the 6-chloro-2-methoxiacridine molecule at either terminus or within the sequence. Acridine has the ability to intercalate efficiently into a double helix. Covalent binding of 6-chloro-2-methoxyacridine therefore increases hybrid stability by providing additional binding energy. Acridine-labeled oligos can thus be used in applications where increased stability of oligonucleotide hybrids is crucial. Adding the dye to the 3’terminus also protects the oligo from exonuclease degradation. Acridine is different from Acridite.
On Acridite: 5'-Acryl labeled oligonucleotides allow the copolymerization of these oligos into polyacrylamide gels. The gel has one or more zones that contain oligos complementary to specific sequences. Running a fragment containing a complementary sequence through such a zone results in trapping of the fragment. These modified oligos find their applications in mutant detection, selecting sequences for cloning, sample preparation and concentration and diagnostic applications.
Thiol modification
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The thiol group is added to the oligo with a C6 spacer. These oligos are left in the 3'-R1-S-S-R2 for shipping so they are more stable. Hence, and prior to use, DTT should be used to cleave the di-Thiol and then desalt appropriately prior to conjugation. The protocol for desalting that we suggest be used is based upon size exclusion and a NAP-10 column from Amersham or other size exclusion column.
The protocol we suggest for cleavage and desalting of the oligo is below:
Re-suspend the oligo in 900 uL of 100 mM Phosphate buffer pH 7.5.
Add 100 uL of .1 M DTT and let sit at room temperature for 1 Hour
Desalt to remove excess DTT and protecting group from oligonucleotide
Prequilibrate a NAP-10* size exclusion column with the buffer of choice
Load 1 mL of sample
Elute with 1.5 mL of customer's preferred buffer
Use immediately for conjugation.
* For more detailed information see the Amersham Biosciences product guide and product instructions.
The Thiol mod structure is: HO-C6-S-S-C6-PO3-oligo
Delivery schedule for custom primers
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荧光素标记抗体技术 (一) 原理 目前用于抗体标记的荧光素主要有异硫氰酸荧光素(Fluorescein isothiocy nate,FITC)或罗达明(Lissamine rhodamine B200, RB200)。在硷性条件下FITC 的碳酰胺键可与抗体赖氨酸的ε氨基共价结合，标记后的抗体仍保持与相应抗原结合的能力。在荧光灯源紫外线或兰紫光激发下产生黄绿色荧光，通过在荧光显微镜下观察或流式细胞仪分析可对相应抗原进行定性、定位或定量的检测。 (二) 操作步骤 将纯化的IgG抗体对PH9～9.5碳酸盐缓冲液透析过夜， 透析后抗体液移入小烧杯中 ↓ 称取适量IFTC，加入二甲亚砜(DMSO)(FITC～1mg/1ml DMSO) 使终浓度为1mgFITC／1mlDMSO FITC／IgG比例:如IgG浓度为1mg／ml，FITC／IgG比例约为50μgFI TC／mgIgG； 如IgG为5～10mg／ml，则比例为25μgFITC／ml IgG 在10ml小烧杯中先放入抗体 ↓ 按上述比例将FITC-DMSO溶液逐滴加入透析后的抗体溶液中 ↓ 将标记物用PBS加至2.5ml，磁力搅拌器室温下避光搅拌2h ↓ 用PD10柱(Sephadex G25柱)除去游离荧光素，先用25ml PBS淋洗G2 5柱 ↓
收集PBS洗脱第一个荧光素结合蛋白峰，测定F／P 比值。第二个荧光素峰为游离荧光素 计算: 2.87×A495 F／P＝──────── A280-0.35×A495 合适的F／P值为2～4。 (三) 试剂器材 1. 纯化的多克隆抗体或单克隆抗体。 2. FITC(Fluorescein-5-Lsothiocyanalte)或其它荧光色素。 3. PBS、DMSO 4. PH9～9.5碳酸盐缓冲液: Na 2CO 3 4.3g，NaHCO 3 8.6g加蒸馏水至500ml。 5. PD10柱(Sephadex G25柱) 6.磁力搅拌器，紫外分光光度计等 (四) 注意事项 1. FITC保存于4℃暗处，使用前待试剂瓶升至室温时开盖称取，以避免潮解。 2. FITC-DMSO液要临用时配制。 3. 碳酸盐缓冲液要新鲜配制。 如有侵权请联系告知删除，感谢你们的配合！
几种常见荧光素极其特性介绍 荧光素（英语：Fluorescein，又称为荧光黄）是一种合成有机化合物，它是具有光致荧光特性的染料，外观为暗橙色/红色粉末，可溶于乙醇，微溶于水，在蓝光或紫外线照射下，发出绿色荧光。荧光染料种类很多，目前常用于标记抗体的荧光素有以下几种：异硫氰酸荧光素，四乙基罗丹明，四甲基异硫氰酸罗丹明，酶作用后产生荧光的物质。目前荧光素广发应用在免疫荧光、免疫荧光染色实验中。 下面介绍几种常用荧光素及其基本生物学特性： 1、异硫氰酸荧光素，简称“FITC”。是一种小分子荧光素，其效率取决于于溶液的pH 值，因此，在使用FITC时应注意溶液的酸碱度。FITC分子量为389.4，最大吸收光波长为490～495nm，最大发射光波长为520～530nm，呈现明亮的黄绿色荧光。 FITC在冷暗干燥处可保存多年，是目前应用最广泛的荧光素。其主要优点是人眼对黄绿色较为敏感，通常切片标本中的绿色荧光少于红色。 2、藻红蛋白，简称“PE”。相对分子质量较大，约为240kD，最大吸收峰为564nm，当使用488nm激光激发时其发射荧光峰值约为576nm，故可能会对其它大探针产生空间位阻。 但PE的化学结构非常稳定，有很高的荧光效率，并易与抗体分子结合。需要注意的是PE作为天然染料，因来源不同可能造成荧光素结构上的微小差别，导致其特征的不一致。 3、PI和EB。两者都具有嵌入到双链DNA和RNA的碱基对中并与碱基对结合的特异性。为了获得特异的DNA分布，染色前必须用RNA酶处理细胞，排除双链RNA的干扰。 PI和EB不能进入完整的细胞膜，因此，又可以用于检测死活细胞。PI和EB各种理化性质相似，但PI比EB的发射光光谱峰向长波方向移动，因而在做DNA和蛋白质双参数测量时，PI的红色荧光和FITC的绿色荧光更易于区分和测量。另外，PI比EB测得的DNA 分布的变异系统（CV值）低，所以PI得到更广泛的应用。
荧光 一、定义 荧光（fluorescence ）又作“萤光”，是指一种光致发光的冷发光现象。当某种常温物质经某种波长的入射光（通常是紫外线或X射线）照射，吸收光能后进入激发态，并且立即退激发并发出比入射光的的波长长的出射光（通常波长在可见光波段）；而且一旦停止入射光，发光现象也随之立即消失。具有这种性质的出射光就被称之为荧光。 二、原理 光照射到某些原子时，光的能量使原子核周围的一些电子由原来的轨道跃迁到了能量更高的轨道，即从基态跃迁到第一激发单线态或第二激发单线态等。第一激发单线态或第二激发单线态等是不稳定的，所以会恢复基态，当电子由第一激发单线态恢复到基态时，能量会以光的形式释放，所以产生荧光。 荧光是物质吸收光照或者其他电磁辐射后发出的光。大多数情况下，发光波长比吸收波长较长，能量更低。但是，当吸收强度较大时，可能发生双光子吸收现象，导致辐射波长短于吸收波长的情况发射。当辐射波长与吸收波长相等时，既是共 荧光强度：荧光强度与该种物质的荧光量子产率、消光系数以及含量等因素有关。荧光量子产率Q：量子产率表示物质将吸收的光能转化为荧光的本领，是荧光物质发出光子数与吸收光子数的比值。荧光蛋白分子的亮度由其量子产率与消光系数的乘积决定，与成像检测灵敏度密切相关。 三、荧光蛋白 1、绿色荧光蛋白（green fluorescent protein,GFP ）
在光谱的绿光区（500nm-525nm）已经发现了多种荧光蛋白，而且来源广泛，包括不同种属的Aequorea 、桡足类动物、文昌鱼以及珊瑚。然而多数有齐聚反应，即使最好的荧光蛋白与EGFP相比，也没有明显的优点。或许目前活细胞成像最好的选择是GFP 衍生的Emerald（祖母绿），它与EGFP的特性相似。Emerald包含F64L 和S65T突变，另外还有四个点突变从而改进了折叠、37℃时的突变率以及亮度。虽然Emerald比EGFP更有效，但含有快速光漂白成分，可能在某些环境下其定量成像会受到影响。 下面主要介绍GFP及其衍生型荧光蛋白： （1）来源绿色荧光蛋白最早由美籍日裔科学家下村修于1962年在水母中发现。这种蛋白质在蓝色波长范围的光照激发下发出绿色荧光，其发光过程需要冷光蛋白质 Aequorin 的帮助，而且，这个冷光蛋白质可与钙离子（Ca2+）相互作用。在水母中发现的野生型绿色荧光蛋白的分子量较小，仅为27~30kDa，而编码GFP的基因序列也很短，为2.6kb 。 （2）性质 GFP由238个氨基酸残基组成。GFP序列中的65-67 位残基（Ser65-Tyr66-Gly67 ）可自发形成荧光发色基团——对羟基苯咪唑啉酮GFP的激发光谱在400nm附近有一个主激发峰，在470nm附近有一个次激发峰。发射光谱在505nm附近有一尖锐的主发射峰，在540nm附近有一肩峰GFP的化学性质相当稳定，无光漂白现象（Photobleaching ），用甲醛固定和石蜡包埋亦不影响其荧光性质。在细胞生物学与分子生物学领域中，绿色荧光蛋白基因常被用作报告基因。 （3）野生型 野生型GFP（wild type GFP, wtGFP ）从一开始就引起了人们极大的兴趣，而且被用作新型的简单报告基因及体内标记，但GFP在异源生物体中的表达并非那么简单。例如，研究人员很早就发现需要在较高的温度下孵育才能在细胞或生物体中表达GFP，并且wtGFP在37℃的热稳定性差。这些都阻碍了它在转基因中的应用。这些难题促使人们进一步筛选分离wtGFP的变体。现在，人们已经找到了荧光强度更强且更耐热的变体。 这些变体多数为经突变的脱辅基蛋白，它们可防止高温导致的错误折叠。近年来出现的新型wtGFP基因突变体的激发和发射谱发生了改变，热稳定性和荧光强度得到了提高，GFP报告基因在小鼠中的应用就是以这些变体作为基础的。 （4）增强型绿色荧光蛋白（EGFP）现在，应用最为广泛的是红移变体增强型GFP （EGFP）。诸如EGFP这些红移变体的最大激发峰发生红向移动，大约为490nm，这一波长也恰好是多数分光设备、流式细胞仪及共聚焦显微镜的常用波长。EGFP有两个氨基酸突变，当被蓝光激发时，它发出的荧光要比wtGFP亮30-40 倍。wtGFP和包括EGFP在内的多数变体半衰期长，所以不适合精确追踪表达的减少或损耗。 （5）不稳定增强型绿色荧光蛋白（dEGFP） 为克服这一问题，人们在1998年构建了不稳定增强型绿色荧光蛋白（dEGFP）。原理就是将EGFP的cDNA融合到小鼠鸟氨酸脱羧酶（Ornithine decarboxylase, ODC）基因的C-末端。ODC含有一个PEST序列，这个序列可促进该蛋白在细胞内的降解。虽然，目前这些不稳定变体还没有在小鼠中应用，但这些变体有利于实时追踪基因表达动力学的研究。 （6）增强型黄色荧光蛋白（EYFP）另一种红移变体是增强型黄色荧光蛋白（EYFP），该变体有四个氨基酸突变。在527nm时，EYFP的发射光从绿色变为黄绿色。EYFP荧光的亮度水平与EGFP相当。EYFP 抗酸性差、对卤化物敏感，使它的应用受到限制。在EYFP 基础上改进的突变体mCitrine[21] 和mVenus[22]是目前应用
同位素示踪与荧光标记技术 [热考解读] 1．同位素示踪法 (1)同位素示踪法：用示踪元素标记的化合物，可以根据这种化合物的放射性，对有关的一系列化学反应进行追踪。这种科学的研究方法叫做同位素示踪法，也叫同位素标记法。(2)应用：可用于研究细胞内的元素或化合物的来源、组成、分布和去向等，进而了解细胞的结构和功能、化学物质的变化、反应机理等。还可用于疾病的诊断和治疗，如碘的放射性同位素可以用来治疗甲状腺肿大。 (3)使用注意事项：一次只能使用一种同位素标记 2．荧光标记法 荧光标记法(Fluorescent Labeling)是利用荧光蛋白或荧光蛋白基因作为标志物对研究对象进行标记的分析方法。 (1)常用的荧光蛋白为绿色和红色两种 ①绿色荧光蛋白(GFP)常用的是来源于发光水母的一种功能独特的蛋白质，分子量为27 kD，具有238个氨基酸，蓝光或近紫外光照射，发射绿色荧光。 ②红色荧光蛋白来源于珊瑚虫，是一种与绿色荧光蛋白同源的荧光蛋白，在紫外光的照射下可发射红色荧光，有着广泛的应用前景。 (2)人教版教材中用到荧光标记法的地方 ①《必修1》P66“细胞融合实验”：这一实验很有力地证明了细胞膜的结构特点是具有一定的流动性。 ②《必修2》P30“基因在染色体上的实验证据”：通过现代分子生物学技术，运用荧光标记的手段，可以很直观地观察到某一基因在染色体上的位置。 (3)荧光标记法特别是在免疫学研究中也有重要的作用，例如免疫荧光抗体标记法。将已知的抗体或抗原分子标记上荧光素，当与其相对应的抗原或抗体起反应时，在形成的复合物上就带有一定量的荧光素，在荧光显微镜下就可以看见发出荧光的抗原抗体结合部位，检测出抗原或抗体。 [命题设计] 1．(2018·山东青岛一模)同位素标记法常用于追踪物质运行和变化规律的研究，下列相关叙述不正确的是() A．给小鼠供应18O2，其呼出气体中可能含有C18O2 B．用含3H标记的尿嘧啶核糖核苷酸的营养液培养洋葱根尖，只能在分生区细胞中检测到放射性 C．用15N标记DNA分子，可用于研究DNA分子的半保留复制 D．用32P标记的噬菌体侵染大肠杆菌，保温、搅拌、离心后可检测到沉淀物中放射性很高
荧光化学传感器是建立在光谱化学和化学波导与量测技术基础上的将分析对象的化学信息以荧光信号表达的传感装置。其主要组成部件有三个(图 1.1)：1．识别结合基团(R),能选择性地与被分析物结合,并使传感器所处的化学环境发生改变。这种结合可以通过配位键，氢键等作用实现。２．信号报告基团(发色团, F），把识别基团与被分析物结合引起的化学环境变化转变为容易观察到的输出信号。信号报告基团起到了信息传输的作用，它把分子水平上发生的化学信息转换成能够为人感知(颜色变化)或仪器检测的信号（荧光等)。3.连接基团(S),将信号报告基团和识别结合基团连接起来,根据设计的不同连接基团可有多种选择，一般用做连接基团的是亚甲基等短链烷基。连接基团的合适与否将直接影响是否有输出信号的产生。信号表达可以是荧光的增强或减弱、光谱的移动、荧光寿命的变化等。 图1．1 荧光探针的结构 1.1.1 荧光探针的一般设计原理 (1) 结合型荧光探针[２1］ +
Analyte Signalling subunit Space Binding subunit Output signal 图1．２ 共价连接型荧光探针 结合型荧光探针是利用化学共价键将识别基团和荧光基团连接起来的一类荧光探针,是比较常见的一类荧光探针。该类探针通过对比加入分析物前后荧光强度的变化、光谱位置的移动或荧光寿命的改变等实现对分析物的检测。在该类荧光化学传感器的设计中,必须充分考虑下列三个方面的因素。(ａ） 受体分子的荧光基团设计、合成:考虑到用于复杂环境体系的荧光检测，要求荧光基团要有强的荧光（高荧光量子产率，有利于提高检测的灵敏性）,Ｓｔｏｋｅs 位移要大（可有效消除常规荧光化合物如荧光素等具有的自猝灭现象)，荧光发射最好要在长波长区(最好位于5０0 nm 以上，可避免复杂体系的常位于短波长区的背景荧光的干扰,另外由于长波长区发射的荧光能量的降低可减少荧光漂白现象的发生而延长传感器的使用寿命）。(b) 受体分子的识别基团：受体分子的识别基团设计以软硬酸碱理论、配位作用以及超分子作用力(如氢键、范德华力等）作为理论指导,多选择含氮、硫、磷杂环化合物作为识别分子。(ｃ) 荧光超分子受体的组装:组装荧光超分子受体就是利用一个连接基将识别基团和荧光基团通过共价键连接在一起,要充分考虑到识别基团和荧光
Please refer to STYLE GUIDE.doc for detailed guidelines Color code legene: Red = Proprietary; Pink = Discontinuation; Green = Anecdotal; Blue = Anything else customers will not see Custom Primers – All Modifications TABLE OF CONTENTS PRODUCT DESCRIPTION SHIPPING CONDITIONS STORAGE CONDITIONS STABILITY QC SPECIFICATIONS PROTOCOL & APPLICATION NOTES Modification/scale/purification Manufacturing Details Fluorescein Rhodamine HEX/TET/FAM Phosphate Biotin Amine Gateway Alexa Other dyes Alkaline Phosphatase Horse Radish Peroxidase Phosphorothioate “Fully-Phosphorothioated" Custom Primers Aldehyde Acridine Thiol Delivery Schedule OligoPerfect Designed Primers 3' Modifications of Oligos Reconstitution Protocol A260/A280 ratio of the oligo Oligo Visualization Troubleshooting “Custom Custom” modifications List of current technology limitations COMPETITOR INFORMATION ALTERNATE PRODUCTS & COMPATIBILITY PRODUCT DOCUMENTATION REFERENCES PRODUCT NAME & CATALOG NUMBER COMPONENTS DISCONTINUATION NOTICE ASSOCIATED PRODUCTS PRODUCT QUALITY ISSUES Returning Primers LICENSING INTERNAL CONTACTS
荧光标记技术在蛋白质定位及功能研究中的应用 Feb 20, 2010No Comments 随着分子生物学、有机化学以及材料科学等学科的进展，最近我们又获得了好几种新型的荧光蛋白标签，这些标签可以用于细胞生物学成像研究。本文将对荧光标志物在蛋白质研究中的优势及劣势进行一番详细的介绍，文章中将重点介绍如何使用荧光标志物研究活体细胞（而不是固定细胞）中的靶蛋白。使用该方法可以对靶蛋白的表达情况、细胞中的定位情况、活性状态等指标进行研究，还将介绍将荧光显微镜与电子显微镜技术相结合的可行性问题。小分子荧光标志物染料、纳米晶体材料，即所谓的“量子点（quantum dots）”材料、自发荧光蛋白、小分子蛋白质标签等等这些材料都可以作为荧光标志物，而且将这几种材料“混合”起来是一种非常有前途的荧光标志物研究新思路。 我们使用荧光技术来研究细胞生物学已经好多年了，而且在从微小的分子层面到完整的有机体层面等各个层面都可以使用荧光技术进行研究。最开始使用的方法是将小分子有机染料与各种抗体相连接，来研究各种目的蛋白。不过这种使用抗体的方法如果需要对细胞内的蛋白质进行研究时，还需要对细胞进行固定和透化操作。因此后来又发展出可以直接在活体细胞内标记某种细胞器、核酸分子或某些离子的荧光标志物。在最近这10年里，荧光蛋白的出现使得进行非侵入性的活体细胞成像成为了可能。使用这种荧光蛋白标志物，我们可以研究目的基因的表达情况，蛋白质运输情况以及各种细胞内动态的生物化学信号通路。使用经过遗传修饰的小分子有机荧光标志物构建的混合系统，我们还可以对蛋白质的寿命进行研究，如果再结合电镜技术和快速光淬灭技术（rapid photoinactivation）还可以对蛋白质的定位情况进行研究。与此同时，半导体纳米晶体材料技术也得到了高度的发展，现在，这种新型的材料在亮度和光稳定性方面都要比传统的荧光标志物好得多，只不过现在这种材料的靶向性还不是很好。本文中我们将对目前荧光标志物及其相关技术的发展进行介绍，同时还将介绍荧光标志物在蛋白质表达、蛋白质活性以及蛋白质功能研究工作中的作用进行介绍。 ?0?2 荧光标志物 小分子有机染料 小分子有机染料是指分子量小于1KD的小分子物质，这种小分子有机染料可以通过与生物大分子共价连接的方式对其进行标记，我们现在对这种染料的最佳检测波长范围、亮度，即吸光系数、光稳定性和自我淬灭特性都有了比较详尽的了解。利用荧光染料的分子策略包括扩展共轭双键、额外添加环状结构增强其刚性、用氟或磺酸盐这类吸电子性的或带电荷的物质进行修饰等。现在市面上已经有数百种这类荧光染料的商业化产品可供选择，而且还在不断增加之中。不过由于这类染料对蛋白质缺乏特异性，因此多与抗体联用（图1A~C）。?0?2 荧光蛋白 第一批用于细胞生物学的荧光蛋白包括藻胆蛋白（phycobiliproteins）和从蓝藻
1光致电子转移(PET) 递给荧光基团的键合基团(RecePtor)，负责光吸收并产生荧光发射信号的荧光基团(Fluorophorc)—其荧光发射强度反映键合基团的结合状态，以及连接键合集团和荧光基团的连接基团(Spacer)。键合基团和荧光基团通常为电子给体或者电子受体。 光致电子转移是指电子给体或电子受体受光激发后，激发态的电子给体与电子受体之间发生电子转移从而导致荧光的淬灭过程。例如，当荧光分子传感器的键合基团是电子给体，荧光基团是电子受体时，具体PET作过程如下:在光激发下，具有电子给予能力的键合基团能够将其处于最高能级的电子转入激发态下荧光基团空出的电子轨道，使被光激发的电子无法直接跃迁巨}到原基态轨道发射荧光，从而导致荧光的淬灭;当键合基团与底物结合后，降低了键合基团的给电子能力，抑制了PET过程，荧光基团中被光激发的电子可以直接跃迁回到原基态轨道，从而增强了的荧光基团的荧光发射。因此在未结合底物前，传感器分子表现为荧光淬灭，一旦键合基团与底物相结合，荧光基团就会发射荧光(见图) 由于与客底物结合前后的荧光强度差别很大，呈现明显的“关”、“开”状态，因此这类荧光化学传感器又被称为荧光分子开关。PET荧光分子传感器的作用机制可由前线轨道理论“来进一步说明(见图 1.5)。
2分子内电荷转移(ICT) ICT荧光化学传感器由推电子基团、吸电子基团通过p电子体系连接而成，在基态时表现为极化结构，一端为缺电子部分，另一端为富电子部分;而在光激发下，偶极矩增大，强化了这种极化特征，容易发 生ICT过程(如图)。 ICT荧光化学传感器的工作原理有两种(见图l.7a):当底物是缺电子基团(阳离子)时，一种是底物与吸电子基团结合，将增大分子内电荷转移程度，导致荧光光谱红移;一种是底物与推电子基团结合，则使原来向共扼体系转移的孤对电子用于与阳离子形成配位键，导致ICT 推一拉电子的特征下降，导致荧光光谱蓝移。当底物是富电子基团(阴离子)时，情况相反。一般情况下，ICT荧光化学传感器对荧光强度的影响不如PET荧光化学传感器显著。典型例子是同时含有吸电子
% %de 非对映体过量百分比（不对称合成术语） %ee 对映体过量百分比（不对称合成术语） A A/MMA 丙烯腈/甲基丙烯酸甲酯共聚物 AA 丙烯酸 AAS 丙烯酸酯-丙烯酸酯-苯乙烯共聚物 ABFN 偶氮（二）甲酰胺 ABN 偶氮（二）异丁腈 ABPS 壬基苯氧基丙烷磺酸钠 Ac 乙酰基 acac 乙酰丙酮基 AIBN 2,2'-二偶氮异丁腈 aq. 水溶液 B BAA 正丁醛苯胺缩合物 BAC 碱式氯化铝 BACN 新型阻燃剂 BAD 双水杨酸双酚A酯 BAL 2，3-巯（基）丙醇 9-BBN 9-硼二环[3.3.1]壬烷 BBP 邻苯二甲酸丁苄酯 BBS N-叔丁基-乙-苯并噻唑次磺酰胺 BC 叶酸 BCD β－环糊精 BCG 苯顺二醇 BCNU 氯化亚硝脲 BD 丁二烯 BE 丙烯酸乳胶外墙涂料 BEE 苯偶姻乙醚 BFRM 硼纤维增强塑料 BG 丁二醇 BGE 反应性稀释剂 BHA 特丁基-4羟基茴香醚 BHT 二丁基羟基甲苯 BINAP （2R,3S）-2.2'-二苯膦－1.1'-联萘，亦简称为联二萘磷，BINAP是日本名古屋大学的Noyori（2001年诺贝尔奖）发展的一类不对称合成催化剂 BL 丁内酯 BLE 丙酮-二苯胺高温缩合物 BLP 粉末涂料流平剂 BMA 甲基丙烯酸丁酯 BMC 团状模塑料 BMU 氨基树脂皮革鞣剂 BN 氮化硼
Bn 苄基 BNE 新型环氧树脂 BNS β－萘磺酸甲醛低缩合物 BOA 己二酸辛苄酯 BOC 叔丁氧羰基（常用于氨基酸氨基的保护）BOP 邻苯二甲酰丁辛酯 BOPP 双轴向聚丙烯 BP 苯甲醇 BPA 双酚A BPBG 邻苯二甲酸丁（乙醇酸乙酯）酯 BPF 双酚F BPMC 2-仲丁基苯基-N-甲基氨基酸酯 BPO 过氧化苯甲酰 BPP 过氧化特戊酸特丁酯 BPPD 过氧化二碳酸二苯氧化酯 BPS 4，4’-硫代双（6-特丁基-3-甲基苯酚）BPTP 聚对苯二甲酸丁二醇酯 Bpy 2,2'-联吡啶 BR 丁二烯橡胶 BRN 青红光硫化黑 BROC 二溴（代）甲酚环氧丙基醚 BS 丁二烯-苯乙烯共聚物 BS-1S 新型密封胶 BSH 苯磺酰肼 BSU N，N’-双（三甲基硅烷）脲 BT 聚丁烯-1热塑性塑料 BTA 苯并三唑 BTX 苯-甲苯-二甲苯混合物 Bu 正丁基 BX 渗透剂 BXA 己二酸二丁基二甘酯 BZ 二正丁基二硫代氨基甲酸锌 Bz 苯甲酰基 C c- 环－ CA 醋酸纤维素 CAB 醋酸-丁酸纤维素 CAM 甲基碳酰胺 CAN 硝酸铈铵 CAN 醋酸-硝酸纤维素 CAP 醋酸-丙酸纤维素 Cat. 催化 CBA 化学发泡剂 CBz 苄氧羰基
关于荧光染料（资料集合） ●人肉眼对光源波长的颜色感觉 红色770-622 nm 橙色622~597 nm 黄色597~577 nm 绿色577~492 nm 蓝靛色492～455nm 紫色455～350nm ●理想的荧光染料一般具有以下几个特点： 1.具有高的光子产量，信号强度高； 2.对激发光有较强的吸收，降低背景信号； 3.激发光谱与发射光谱之间距离较大，减少背景信号的干扰； 4.易与被标记的抗原、抗体或其他生物物质结合而不影响被标记物的特异性； 5.稳定性好，不易受光、温度、PH、标本抗凝剂和固定剂的影响。 ●染料在生物化学中最早的应用是直接对切片进行染色，然后进行观察。随着生物技术、计算机技术以及荧光光谱测定技术的不断发展，许多染料尤其是荧光染料在细胞检测、肿瘤基因蛋白分析、毒物分析、临床医疗诊断等方面得到了广泛的应用。 荧光染料泛指吸收某一波长的光波后能发射出另一大于吸收光波长的光波的物质。利用荧光染料进行抗体标记分析在现代生物免疫学领域中应用广泛，并逐步显示出明显的优越性。 下面简要介绍应用于标记抗体的荧光染料及其种类： 1.荧光素类染料，包括异硫氰酸荧光素(FITC)、羟基荧光素(FAM)、四氯荧光素(TET)等及其类似物。这是一类具有较多苯环的化合物。应用最广泛的是FITC(如图为FITC标记的组织荧光图)，在488nm 处由氩离子激光激发，发射525nm的蓝绿色荧光。FITC能够与各种抗体蛋白结合，并在碱性溶液中稳定呈现蓝绿色荧光。 2.罗丹明类染料，包括红色罗丹明（RBITC）、四甲基罗丹明（TAMRA）、罗丹明B（TRITC）等。TRITC在550nm处被激发可发射出570nm的黄色荧光。 3.Cy系列菁染料，菁染料通常有两个杂环体系组成，包括Cy2、Cy3、Cy3B、Cy3.5、Cy5、Cy5.5、Cy7及其类似物。 4.Alexa系列染料，它是由MolecularProbes开发的系列荧光染料。其激发光和发射光光谱覆盖大部分可见光和部分红外线光谱区域，应用广泛。以高亮度、稳定性、仪器兼容性、多种颜色、pH值不敏
绿色萤光蛋白(green fluorescent protein)，简称GFP，这种蛋白质最早在一种学名Aequorea victoria的水母中发现。其基因所产生的蛋白质，在蓝色波长范围的光线激发下，会发出绿色萤光。这个发光的过程中还需要冷光蛋白质Aequorin的帮助，且这个冷光蛋白质与钙离子(Ca+2)可产生交互作用。 由水母Aequorea victoria中发现的野生型绿色萤光蛋白，395nm和475nm分别是最大和次大的激发波长，它的发射波长的峰点是在509nm，在可见光绿光的范围下是较弱的位置。由海肾(sea pansy)所得的绿色萤光蛋白，仅有在498nm有一个较高的激发峰点。 在细胞生物学与分子生物学领域中，绿色萤光蛋白基因常被用作为一个报导基因(reporter gene)。一些经修饰过的型式可作为生物探针，绿色萤光蛋白基因也可以克隆到脊椎动物(例如:兔子上进行表现，并拿来映证某种假设的实验方法。 我们这边细胞组的基本上都在用这个东东。标记细胞 GFP的分子结构和发光机制 绿色荧光蛋白为一个由238个氨基酸残基组成的单链，GFP有两个吸收峰，主峰在395nm，次峰在470nm，其荧光发射峰在509nm。GFP 的化学性质相当稳定，其变性需要在90℃或pH<4或pH>12的条件下用6mollL盐酸胍处理，这一性质与GFP的结构特性相关。 Yang等的研究表明，GFP是由两个相当规则的内含一个α-螺旋和外面包围l1个β-折叠的β-桶状结构组成的二聚体，β-桶状结构直径约3nm，高约4nm。β折叠彼此紧密结合，象桶板一样形成桶状结构的外围，并且形成了一个规则的氢键带。桶状结构和位于其末端的短α螺旋以及环状结构一起组成一个单独的致密结构域，没有可供扩散的配体进入缝隙。这种坚实的结构保证了其稳定和抗热、抗变性的特点。 GFP的生色基团附着于α-螺旋上，几乎完美的包被于桶状结构中心。位于圆桶中央的α-螺旋含有一个由六肽组成的发光中心，而发光团是由其中的三肽Ser65-Tyr66-Gly67经过环化形成了对羟基苯咪唑啉酮。GFP的生色基团是蛋白质自身催化环化的结果，环化是一个有氧过程，在严格厌氧条件下GFP不能形成荧光，因为GFP的生色团形成需要O2使Tyr66脱氢氧化。生色基团通过Tyr66的脱质子(酚盐)和质子化状态(羟酚基)的转换决定荧光发射，此模型为Yang等的晶体学证据所支持。 GFP在生物技术中的应用研究 1．分子标记 作为一种新型的报告基因，GFP已在生物学的许多研究领域得到应用。利用绿色荧光蛋白独特的发光机制，可将GFP作为蛋白质标签(protein tagging)，即利用DNA重组技术，将目的基因与GFP基因构成融合基因，转染合适的细胞进行表达，然后借助荧光显微镜便可对标记的蛋白质进行细胞内活体观察。由于GFP相对较小，只有238个氨基酸，将其与其他蛋白融合后不影响自身的发光功能，利用GFP的这一特性已经加深了我们对细胞内一些过程的了解，如细胞分裂、染色体复制和分裂，发育和信号转导等。1996年，Ehrdardt等人首次报道了利用GFP的特性研究细胞分化蛋白FtsZ的定位。研究显示FtsZ在细胞分裂位点形成了一个环状物，且至少有9种蛋白在细胞分裂中起重要作用，尽管对这些蛋白功能仍然不是很清楚，但是利用GFP融合蛋白已经搞清楚了它们聚合的顺序以及在蛋白定位中的一些特征。利用GFP来检测目标蛋白的定位已为我们提供了一种对细胞内的一些基本的生理过程进行更详尽观察的新方法。 除用于特定蛋白的标记定位外，GFP亦大量用于各种细胞器的标记如细胞骨架、质膜、细胞核等等。Shi等人曾报道将GFP融合到大肠杆菌细胞膜表面用作标记蛋白，这一技术将有助于提高多肽库的筛选效率、疫苗的研制、构建细胞生物传感器用作环境检测以及探测信号转导过程等等。这些都为传统生物学研究提供了新思路和新方法，成为交叉学科研究的热点。 2．药物筛选 许多新发展的光学分析方法已经开始利用活体细胞来进行药物筛选，这一技术能从数量众多的化合物中快速筛选出我们所感兴趣的药物。基于细胞的荧光分析可分为三类：即根据荧光的密度变化、能量转移或荧光探针的分布来研究目标蛋白如受体、离子通道或酶的状态的变化。荧光探针分布是利用信号传导中信号分子的迁移功能，将一荧光蛋白与信号分子相偶联，根据荧光蛋白的分布情况即可推断信号分子的迁移状况，并推断该分子在迁移中的功能。由于GFP分子量小，在活细胞内可溶且对细胞毒性较小，因而常用作荧光探针。 在细胞体内分子之间的相互作用非常复杂，其中很多涉及到信号分子在细胞器之间的迁移。例如当信号分子和某一特殊受体结合后常会导致配体-受体复合物从某一细胞区域迁移到另一区域，而这一迁移过程通常会介导一重要的生理功能。因而，这些受体常常被用作药物筛选的目标，若某一药物具有与信号分子类似的功能，那么该药物即具有潜在的医药价值。利用GFP荧光探针，将很容易从数量众多的化合物中判断出那些化合物具有与信号分子相似的能引起配体一受体复合物迁移并介导生理反应的功能，且这一筛选过程简单方便，所需成本也很低。利用这一原理，已经成功构建了一个筛选模型用于研究药物介导的糖皮质激素受体(hGR)的迁移过程。在一96孔板中培养细胞，并以一编码hGR GFP蛋白的质粒转染该细胞。当细胞用待筛选的药物处理后，hGR-GFP从细胞质迁移人细胞核的过程可实时或在某一时段
https://www.doczj.com/doc/0c17643869.html,/?p=21977 首页专题译述会议展览技术方法教学视频热点话题生命百态研究前沿科研综述电子杂志RSS 订阅当前位置: 生命奥秘> 技术方法> 文章正文荧光标记技术在蛋白质定位及功能研究中的应用 cyq 发表于2010-02-20 14:48 | 来源：| 阅读 随着分子生物学、有机化学以及材料科学等学科的进展，最近我们又获得了好几种新型的荧光蛋白标签，这些标签可以用于细胞生物学成像研究。本文将对荧光标志物在蛋白质研究中的优势及劣势进行一番详细的介绍，文章中将重点介绍如何使用荧光标志物研究活体细胞（而不是固定细胞）中的靶蛋白。使用该方法可以对靶蛋白的表达情况、细胞中的定位情况、活性状态等指标进行研究，还将介绍将荧光显微镜与电子显微镜技术相结合的可行性问题。小分子荧光标志物染料、纳米晶体材料，即所谓的“量子点（quantum dots）”材料、自发荧光蛋白、小分子蛋白质标签等等这些材料都可以作为荧光标志物，而且将这几种材料“混合”起来是一种非常有前途的荧光标志物研究新思路。 我们使用荧光技术来研究细胞生物学已经好多年了，而且在从微小的分子层面到完整的有机体层面等各个层面都可以使用荧光技术进行研究。最开始使用的方法是将小分子有机染料与各种抗体相连接，来研究各种目的蛋白。不过这种使用抗体的方法如果需要对细胞内的蛋白质进行研究时，还需要对细胞进行固定和透化操作。因此后来又发展出可以直接在活体细胞内标记某种细胞器、核酸分子或某些离子的荧光标志物。在最近这10年里，荧光蛋白的出现使得进行非侵入性的活体细胞成像成为了可能。使用这种荧光蛋白标志物，我们可以研究目的基因的表达情况，蛋白质运输情况以及各种细胞内动态的生物化学信号通路。使用经过遗传修饰的小分子有机荧光标志物构建的混合系统，我们还可以对蛋白质的寿命进行研究，如果再结合电镜技术和快速光淬灭技术（rapid photoinactivation）还可以对蛋白质的定位情况进行研究。与此同时，半导体纳米晶体材料技术也得到了高度的发展，现在，这种新型的材料在亮度和光稳定性方面都要比传统的荧光标志物好得多，只不过现在这种材料的靶向性还不是很好。本文中我们将对目前荧光标志物及其相关技术的发展进行介绍，同时还将介绍荧光标志物在蛋白质表达、蛋白质活性以及蛋白质功能研究工作中的作用进行介绍。 荧光标志物 小分子有机染料 小分子有机染料是指分子量小于1KD的小分子物质，这种小分子有机染料可以通过与生物大分子共价连接的方式对其进行标记，我们现在对这种染料的最佳检测波长范围、亮度，即吸光系数、光稳定性和自我淬灭特性都有了比较详尽的了解。利用荧光染料的分子策略包括扩展共轭双键、额外添加环状结构增强其刚性、用氟或磺酸盐这类吸电子性的或带电荷的物质进行修饰等。现在市面上已经有数百种这类荧光染料的商业化产品可供选择，而且还在不
分子生物学实验设计报告 绿色荧光蛋白的克隆表达 ——闵霞（2013141241165）李彩云（2013141241095） 一、引言 基因标记技术是近年来发展起来的分子生物学技术。荧光蛋白基因在标记基因方面由于具有独特的优点而广受科学家们的关注。荧光蛋白是海洋生物体内的一类发光蛋白，分为绿色荧光蛋白、蓝色荧光蛋白、黄色荧光蛋白和红色荧光蛋白。 绿色荧光蛋白（green fluorescent protein，GFP）是一类存在于包括水母、水螅和珊瑚等腔肠动物体内的生物发光蛋白。当受到紫外或蓝光激发时，GFP发射绿色荧光。它产生荧光无需底物或辅因子，发色团是其蛋白质一级序列固有的。 基因克隆技术包括把来自不同物的基因同有自主复制能力的载体DNA在体外人工连接，构建成新的重组DNA，然后送入受体生物中去表达，从而产生遗传物质和状态的转移和重新组合。采用重组DNA技术，将不同来源的DNA分子在体外进行特异性切割，重新连接，组装成一个新的杂合DNA 分子。在此基础上，这个杂合分子能够在一定的宿主细胞中进行扩增，形成大量的子代分子。 本次实验中，分子克隆质粒载体所携带的外源基因是EGFP绿色荧光蛋白，实验的最终目的是将EGFP基因插入表达载体pET-28a中，组成重组子，并导入到大肠杆菌细胞中并诱导其表达，培养出绿色的大肠杆菌菌落。为此，我们要利用碱变性法将大肠杆菌中的质粒DNA提取出来，并通过Bam HI和NotⅠ两种酶的双酶切作用，从而获得目的外源基因片段EGFP和表达载体pET-28a质粒的DNA，然后通过连接酶连接后形成重组子，并通过氯化钙法导入大肠杆菌感受态细胞中，让其在含有Amp和IPTG的LB琼脂平板上生长繁殖，最后通过观察大肠杆菌能否在含有Amp和IPTG的LB平板上长出绿色的菌落，来判断EGFP基因工程菌的构建效果 二、主要路线： 1、质粒DNA的提取 2、琼脂糖凝胶电泳检测质粒DNA 3、酶切连接重组质粒 4、重组质粒的扩增 5、菌落PCR法鉴定阳性克隆 6、目的荧光蛋白基因的表达 1、质粒DNA的提取 实验原理： 1）质粒是一种染色体外的遗传因子，大小在1kb~200kb之间，是具有双链闭合环状结构的DNA分子，主要发现于细菌、放线菌和真菌细胞中。质粒具有自主复制能力，，能使子代保持他们恒定的复制数，可表达它携带的遗传信息。它可以独立游离于细胞质内，也可以整合到细菌染色体中，它离开宿主细胞就不能复制，而它控制的许多生物学功能也是对宿主细胞
荧光素FITC标记抗体的方法 当FITC在碱性溶液中与抗体蛋白反应时，主要是蛋白质上赖氨酸的r氨基与荧光素的硫碳胺键(thiocarbmide)结合，形成FITC-蛋白质结合物，即荧光抗体或荧光结合物。一个IgG分子中有86个赖氨酸残基，一般最多能结合15～20个，一个IgG分子可结合2～8个分子的FITC，其反应式如下 FITC-N=C=S + N-H2-蛋白质→ FITC-NS-C-N-H2-蛋白质 常用Marsshall(1958)法标记荧光抗体，也可以根据条件采用Chadwick等标记法或Clark等(1963)的透析标记法。 1．Marsshall法 (1)材料抗体球蛋白溶液、0．5mol／L(pH9．0)碳酸盐缓冲液、无菌生理盐水、异硫氰酸荧光 素、1％硫柳汞水溶液、50ml小烧杯、4℃冰箱、电磁搅拌器、透析袋、玻棒、pH7．2或 3．0的0．01mol／LPBS等。 (2)方法及步骤①抗体的准备取适量已知浓度的球蛋白溶液于烧杯中，再加人生理盐水及碳酸盐缓冲液，使最后免疫球蛋白浓度为
20mg／ml，碳酸盐缓冲液容量为总量的1／10，混匀，将烧瓴置电磁搅拌器上(速度适当以不起泡沫为宜)5～10min。 ②荧光素的准备根据欲标记的蛋白质总量，按每毫克免疫球蛋白加0．01mg荧光色素，用分析天平准确称取所需的异硫氰酸荧光素粉末。也可用下述公式计算出免疫球蛋白、荧光素的量，还可以算出需加缓冲液的量。 a．蛋白溶液：含量Amg／m1；容积Bml。 b．总蛋白量(AXB)＝Crag。 c．C／20～C／10＝Dmg(如蛋白含量低于20mg／ml，用C／10；如高于20mg／ml，用C／20)。 d．荧光素FITC的量：(1／50～2／100)XC＝Emg。 e．巳0．5mol／L(pH9．5)碳酸盐缓冲液D／10＝Fml。 f．PBS量D-(B+F)=Gml。 注：A为蛋白含量，mg／ml；B为蛋白质溶液的容积；C为蛋白总量，mg；D为常数，mg；正为荧光素的量，mg；F为碳酸盐缓冲液的容积，ml；G为PBS的容积，ml。 ③结合(或标记) 边搅拌边将称取的荧光色素渐渐加入球蛋白溶液中，避免将荧光素粘于烧瓶壁(大约在5—10min内加完)，加完后，
荧光染料基础知识大全 益阳纺织染整团队今天 荧光显微镜技术的基本原理是借助荧光剂让细胞成分呈现高度具体的可视化效果，比如在目的蛋白后面连一个通用的荧光蛋白—GFP。在组织样本中，目的基因无法进行克隆，则需要用免疫荧光染色等其他技术手段来观察目的蛋白。为此，就需要利用抗体，这些抗体连接各种不同的荧光染料，直接或间接地与相应的靶结构相结合。此外，借助荧光染料，荧光显微镜技术不只局限于蛋白质，它还可以对核酸、聚糖等其他结构进行染色，即便钙离子等非生物物质也可以检测出来。 1免疫荧光 (IF) 在荧光显微镜技术中，可以通过两种方式观察到你的目的蛋白：利用内源荧光信号，即通过克隆手段，用遗传学方法将荧光蛋白与目的蛋白相连；或利用荧光标记的抗体特异性结合目的蛋白。 有些生物学问题采用第二种方法会更有用或更有必要。比如，组织学样品无法使用荧光蛋白，因为通常来说，标本都是从无法保存荧光蛋白的生物体中获取。此外，当有一个有功能的抗体可用时，免疫荧光法会比荧光蛋白技术快很多，因为后者必须先克隆目的基因再将DNA转染到适当的细胞中。 荧光蛋白的另一项劣势在于其本身属于蛋白质。因此，细胞内的这些荧光蛋白具有特定的蛋白质特性，其会导致附着的目的蛋白质发生功能紊乱或出现误释的情况。然而，荧光蛋白技术仍然是观察活细胞的首选方法。 免疫荧光法利用了抗体可以和相应抗原特异性结合的这个特性，对此它还有两种不同的表现形式。最简单的方式是使用可与目的蛋白相结合的荧光标记抗体。这种方法被称为“直接免疫荧光法”。 在很多情况下，我们可以利用两种不同特性的抗体。第一种抗体可以结合目的蛋白，但其本身并未进行荧光标记（一抗）。第二种抗体本身就携带荧光染料（二抗），并且可以特异性结合一抗。这种方法被称为“间接免疫荧光法”。 这种方法存在诸多优势。一方面，它会产生放大效应，因为不只一个二抗可以与一抗相结合。另一方面，没有必要始终用荧光染料标记目的蛋白的每个抗体，但可以使用市售荧光标记的二抗。免疫荧光中广泛使用的荧光染料包括FITC、TRITC 或一些Alexa Fluor?染料，下文均有提及。 2FITC 和TRITC 异硫氰酸荧光素(FITC) 是一种有机荧光染料，目前，这种荧光染料仍用于免疫荧光和流式细胞术中。在495/517 nm 处，该染料会产生激发/发射峰值，并可借助异硫氰酸盐反应基团与不同抗体结合，该基团可以和蛋白质上的氨基、巯基、咪唑、酪氨酰、羰基等基团相结合。 而它的基本成分——荧光素，其摩尔质量为332 g/mol，常被用作荧光示踪剂。FITC(389 g/mol) 是用于荧光显微镜技术的首批染料，且其被当成Alexa Fluor?488 等后续荧光染料的发端。该染料的荧光活性取决于它的大共轭芳香电子系统，而该系统受蓝色光谱中的光所激发。
1光致电子转移(PET) 光致电子转移是指电子给体或电子受体受光激发后，激发态的电子给体与电子受体之间发生电子转移从而导致荧光的淬灭过程。具体PET过程如下:在光激发下，具有电子给予能力的键合基团能够将其处于最高能级的电子转入激发态下荧光基团空出的电子轨道，使被光激发的电子无法直接跃迁到原基态轨道发射荧光，从而导致荧光的淬灭;当键合基团与底物结合后，降低了键合基团的给电子能力，抑制了PET过程，荧光基团中被光激发的电子可以直接跃迁回到原基态轨道，从而增强了的荧光基团的荧光发射。因此在未结合底物前，传感器分子表现为荧光淬灭，一旦键合基团与底物相结合，荧光基团就会发射荧光(见图) 由于与客底物结合前后的荧光强度差别很大，呈现明显的“关”、“开”状态，因此这类荧光化学传感器又被称为荧光分子开关。PET荧光分子传感器的作用机制可由前线轨道理论来进一步说明(见图1.5)。 2002年Nolan 小组合成了手性的二氮杂环-9-冠-3 衍生物化合物1，它是第一个用来检测Li+的PET 荧光探针[56]。在乙腈溶液中，相较于其它碱金属和碱土金属，能够高选择性的识别锂离子。用280 nm 光激发，不断向溶液中加入
LiClO4，化合物 1（Φ = 0.022）对Li+的滴定表现出 5 倍荧光信号增强效应，表明从胺的冠醚到荧光团的电子转移，荧光量子效率升高（Φ = 0.11），形成 1 : 1 的配合物，结合常数 log β = 5.4。 Gunnlaugsson, Bichell, Nolan, A Novel Fluorescent Photoinduced Electron Transfer (PET) Sensor for Lithium [J]. Tetrahedron Lett., 2002, 43, 4989-4992. NH H 3C O HN CH 3 O N O N ×× PET PET Li + Bozdemir, Altan Sozmen, Fazli Buyukcakir, et al. Reaction-Based Sensing of Fluoride Ions Using Built-in Triggers for Intramolecular Charge Transfer and Photoinduced Electron Transfer[J]. Organic Letters, 2010, 12(7) : 1400-1403. 2010年Akkaya 等[18]通过在BODIPY 的中位引入一个含三异丙基硅烷的酚盐基团,已知酚盐是强的给电子基团,当被硅烧保护后,酚盐的强给电子能力被抑制,即PET 现象被抑制,所以探针2在与F 离子作用之前发出很强的荧光,当探针与F-离子作用之后,硅浣保护基团被去除,酚盐的强给电子能力恢复,发生PET 现象,荧光被淬灭。在F-离子的浓度达到0.5mM 时,探针的荧光被完全淬灭。