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PARTS GUIDE MANUAL APRIL 2005bizhub 162INFORMATION FOR PARTS GUIDE MANUALTo find correct Parts No., refer to the “HOW TO MAKE THE BEST USE OF THIS MANUAL” in the following page.HOW TO MAKE THE BEST USE OF THIS MANUAL1When you order, please check the proper figures beforehand that are on Our Parts Guide Manual, and order with the appropriate figures.2For screws, Nuts, Washers, retaining rings and Pins which are used in this model, one letter is shown on the Standard parts column of Parts list and exploded diagrams.3In order to maintain safety of the product, some specific parts composed of this product are set up as "essential safety parts".4The assigned parts number for the "essential safety parts" is indicated as "SP00-****".When replacing these parts, follow precautions for disassembling and installing which are listed in the Service Manual.Do not use any parts that are not set up as5 means that there are exclusive parts for each destination.Please check the appropriate destination when you order.6Revision MarkMarked as on the illustration shows that the revision has been made. 7All rights reserved. (any reprints or quotations are prohibited.)Use of this parts guide manual should be strictly supervised to avoid dis-closure of confidential information.パーツガイドマニュアルのご案内サービス部品をご発注の際には、下記に示す “パーツガイドマニュアルの活用にあたって”をご参照の上、正しい部品番号にてお願い致します。
MSD GOLD™ SULFO-TAG NHS-EsterMSD GOLD SULFO-TAG™ NHS-Ester 150 nmol (sufficient for conjugating 1 mg of IgG) R91AO-12 µmol (sufficient for conjugating 10 mg of IgG) R91AO-2MSD GOLD SULFO-TAG NHS-Ester Conjugation Packs Pack 1 (sufficient for conjugating 5 x 200 µg of IgG) R31AA-1 Pack 2 (sufficient for conjugating 5 x 1 mg of IgG) R31AA-2MSD Labeling ReagentsMSD GOLD SULFO-TAG NHS-EsterFor labeling aminesNote:150 nmol size of MSD GOLD SULFO-TAG NHS-Ester is sufficient for conjugating 1 mg of IgG and the 2 µmol size is sufficient for conjugating 10 mg of IgG at a challenge ratio of 20.Each MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack contains enough material for 5 reactions. At a challenge ratio of 20, Pack 1 is sufficient for conjugating 200 µg of IgG per reaction, and Pack 2 is sufficient for conjugating 1 mg of IgG per reaction.This package insert must be read in its entirety before using this product.FOR RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURES.MESO SCALE DISCOVERY®A division of Meso Scale Diagnostics, LLC.1601 Research BoulevardRockville, MD 20850-3173 USAMESO SCALE DISCOVERY, MESO SCALE DIAGNOSTICS, MSD, MSD GOLD, DISCOVERY WORKBENCH, MULTI-ARRAY, MULTI-SPOT, QUICKPLEX, SECTOR, SECTOR PR, SECTOR HTS, SULFO-TAG, U-PLEX, V-PLEX, S-PLEX, STREPTAVIDIN GOLD, MESO, , SMALL SPOT (design), 96 WELL 1, 4, 7, & 10-SPOT (designs), 384 WELL 1 & 4-SPOT (designs), MSD (design), U-PLEX (design), V-PLEX (design), S-PLEX (design), and SPOT THE DIFFERENCE are trademarks and/or service marks of Meso Scale Diagnostics, LLC. ©2015 Me s o Scale Diagnostics, LLC. All rights reservedTable of ContentsIntroduction (4)Preparation of MSD GOLD SULFO-TAG Conjugates (5)MSD GOLD SULFO-TAG NHS-Ester Conjugation Packs (6)Additional Materials and Equipment (6)Protocol (8)Storage, Handling, and Stability (10)FAQs (11)Worksheet (14)Ordering InformationMSD Customer ServicePhone: 1-240-314-2795Fax: 1-301-990-2776Email: CustomerService@MSD Scientific SupportPhone: 1-240-314-2798Fax: 1-240-632-2219 attn: Scientific SupportEmail: ScientificSupport@IntroductionThis protocol details the conjugation procedure for proteins of molecular weight (MW) > 40,000 D altons using MSD GOLD SULFO-TAG NHS-Ester label. The straightforward procedure involves an optional buffer exchange step, a 2-hour incubation step, and a mandatory buffer exchange step to quickly isolate the conjugated protein using a spin column. MSD GOLD SULFO-TAG NHS-Ester (Figure 1) is an amine reactive, N-hydroxysuccinimide ester which readily couples to primary amine groups of proteins under mildly basic conditions to form a stable amide bond.MSD GOLD SULFO-TAG conjugates are stable and may be used at low concentrations. These features minimize time, cost, and labor as large batches of a stable conjugate can be prepared, validated, and used for long periods of time. Its excellent performance characteristics and simple conjugation procedure make MSD GOLD SULFO-TAG NHS-Ester the product of choice for molecules that contain primary amines (e.g., lysine-containing proteins). MSD GOLD SULFO-TAG offers low non-specific binding, resulting in highly sensitive detection when used in conjunction with MSD instruments.Figure 1: MSD GOLD SULFO-TAG NHS-EsterPreparation of MSD GOLD SULFO-TAG ConjugatesGeneral NotesIn order to minimize hydrolysis of MSD GOLD SULFO-TAG NHS-Ester, the reagent should be dissolved in cold distilled water just prior to its addition to the protein solution. If necessary, the stock MSD GOLD SULFO-TAG NHS-Ester solution can be kept on ice for up to 10 minutes. The reconstituted solution is unstable and any unused material should be discarded. Consider conjugating more than one protein at the same time to maximize the use of the MSD GOLD SULFO-TAG NHS-Ester reagent. Generally, 150 nmol of MSD GOLD SULFO-TAG NHS-Ester is sufficient for conjugation of up to 1 mg of IgG.The labeling ratio is the number of molecules of MSD GOLD SULFO-TAG conjugated to each molecule of protein. Optimal conjugation ratios for a MSD GOLD SULFO-TAG conjugated protein should be determined empirically for each specific application. For most applications using IgG antibodies (MW ~150,000), optimal performance is obtained with conjugation ratios between 2:1 and 20:1. In this range, assay signals typically show a linear dependence on conjugation ratio. Conjugation ratios significantly higher than 10:1 can be counterproductive and may lead to elevated background signals or loss of binding activity. For proteins that are significantly smaller than IgGs, lower conjugation ratios (between 1:1 and 5:1) may provide better assay performance.The challenge ratio is the number of moles of MSD GOLD SULFO-TAG per mole of protein in the conjugation reaction mixture. The challenge ratio required to achieve a specific conjugation ratio depends on a number of factors including pH, temperature, protein concentration, protein size, and the number of lysines available for coupling. Conjugating a 2 mg/mL IgG solution using the standard conditions described in this protocol will typically result in a label incorporation of approximately 50% (i.e., a challenge ratio of 10:1 will result in a conjugation ratio of about 5:1). Conjugation efficiencies for other proteins may be different. In general, conjugating with high protein concentrations of 1–2 mg/mL in slightly alkaline PBS (pH 7.9) in the absence of preservatives yields the best conjugation efficiencies. Maintaining consistent conjugation conditions (protein concentration, buffer type, MSD GOLD SULFO-TAG NHS-Ester concentration, incubation time, and temperature) is important when preparing multiple batches of conjugated protein in order to achieve consistent assay results.When developing immunoassays, MSD recommends conjugating antibodies using the standard conditions outlined in this document and challenge ratios of 6:1, 12:1, and 20:1 to identify optimal conjugation conditions. If evaluating different conditions is not possible due to limited reagent quantities, a challenge ratio of 20:1 will generally provide good performance. For immunogenicity applications where an antibody drug or protein therapeutic is used, the suggested challenge ratios are 12:1 and 6:1 MSD GOLD SULFO-TAG:drug. If only one ratio is tested, a 10:1 challenge ratio is recommended. For details on building immunogenicity assays, please refer to the Bridging Immunogenicity Guidelines for Assay Development at . The protocol describes the MSD GOLD SULFO-TAG conjugation procedure for proteins with a MW > 40,000 D a. Smaller proteins/polypeptides may also be conjugated using MSD GOLD SULFO-TAG NHS-Ester as long as they have an accessible lysine or the N-terminal amino group; however, alternative separation methods may be needed to remove unconjugated label. MSD offers a variety of services for the custom conjugation of reagents including proteins, peptides, and non-proteinaceous molecules.MSD GOLD SULFO-TAG NHS-Ester Conjugation PacksMSD offers all-inclusive conjugation packs that include the components and guidance that may be necessary for conjugating and purifying detection reagents with MSD GOLD SULFO-TAG label. Two sizes of conjugation packs are offered: MSD GOLD SULFO-TAG Conjugation Pack 1 and Pack 2. The two packs enable the conjugation of different amounts of IgG. MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack 1 contains materials for conjugation and purification of up to 200 µg of IgG per reaction, and MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack 2 allows conjugation of up to 1 mg of IgG per reaction. Each pack contains enough material for 5 reactions (i.e., 5 vials of MSD GOLD SULFO-TAG NHS-Ester).Table 1. Components of MSD GOLD SULFO-TAG Conjugation Packs*MSD GOLD SULFO-TAG Conjugation Pack 1 includes 10 columns of 0.5 mL capacity and 10 syringes of 1 mL size, and MSD GOLD SULFO-TAG Conjugation Pack 2 includes 10 columns of 5 mL capacity and 10 syringes of 3 mL size.**The pH of Conjugation Buffer is stable for up to 2 weeks after opening the bottle. For long term use, it is recommended to readjust the pH of the solution.Additional Materials and EquipmentThe following additional materials may be required. Some items (denoted with a *) are included in the MSD GOLD SULFO-TAG Conjugation Packs.1.Conjugation Buffer* (Phosphate-buffered saline (PBS), pH 7.9, preservative-free)2.Conjugate Storage Buffer* (PBS pH 7.4 + 0.05% Sodium Azide)3.Polypropylene microfuge tubes4.Spin columns.* MSD recommends the use of Zeba Spin Desalting Columns, 40K MWCO of various sizes from ThermoScientific, Catalog # 87766–877735.15 mL conical tubes for use with Zeba Spin Desalting Columns, 40K MWCO, 2 mL column size6.Protein assay such as BCA, Bradford, or Lowry7.MSD Blocker A (optional), Catalog # R93AA-2 (250 mL) and R93AA-1 (1 L)8.Spectrophotometer capable of an OD455 measurementReagent Storage Size Quantity DescriptionMSD GOLD SULFO-TAG NHS-Ester ≤-70°C 150 nmol 5 vials MSD GOLD SULFO-TAG NHS-Ester label forcoupling to antibodies and other proteinsZeba Spin Desalting Column, 40K MWCO* 2–8°C 0.5 mL or5 mL10 columns Size exclusion chromatography columns for thepurification of proteins larger than 40,000 DaFilter, 0.22 µm RT N/A 10 each Filter for use during purification Syringe * RT N/A 10 each Syringe for use during purificationConjugation Buffer** 2–8°C 40 mL 1 bottle Phosphate-buffered saline (PBS), pH 7.9, preservative-freeConjugate Storage Buffer RT 40 mL 1 bottle PBS pH 7.4 + 0.05% Sodium Azide9. 0.2 µm filter* (optional) Table 2. Suggestions for 0.2 µm filter10. Concentrator (optional) Table 3. Suggestions for concentratorsNoteThe following table lists the catalog numbers of the Zeba Spin Desalting Columns, 40K MWCO, from Thermo Scientific and the recommended sample volume for each column.Table 4. Catalog numbers of Zeba Spin Desalting Columns from Thermo ScientificVendorCatalog # Volume of Conjugated ProteinWhatmanAV125EAQU ≥ 2.0 mL Millipore or Fisher Scientific (MILLEX-GV)SLGV004SL0.2–1.0 mLVendorCatalog # Range Millipore BIOMAX-50 concentrator, 50 MWCO UFV5BQK25 0.05–0.5 mL AMICON Ultra-4 concentrator, PLQK Ultracel-PL Membrane, 50 MWCOUFC805008 0.5–4.0 mL AMICON Ultra-15 concentrator, PLQK Ultracel-PL Membrane, 50 MWCOUFC9050240.5–15.0 mLThermo ScientificCatalog #Number of Columns/packZEBA Spin Desalting Column VolumeRecommended Volume of the Conjugation Reaction87766 25 0.5 mL 70–100 µL 87767 50 0.5 mL 70–100 µL 87768 5 2 mL 200–450 µL 87769 25 2 mL 200–450 µL 87770 5 5 mL 300–1,000 µL 87771 25 5 mL 300–1,000 µL 87772 5 10 mL 1,000–2,000 µL 877732510 mL1,000–2,000 µLProtocol1.Prepare a 1–2 mg/mL solution of the protein to be conjugated in Conjugation Buffer. Antibodies in a storage buffer withpreservatives such as sodium azide or EDTA must be buffer exchanged before the conjugation reaction. It is recommended that dilute protein solutions be concentrated to at least 1 mg/mL. Protein solutions should be concentrated and/or buffer exchanged using the spin columns described above or an alternative centrifugal filtration/concentration unit that has been equilibrated with preservative-free PBS, pH 7.9. (Use Conjugation Buffer when using MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack.) Filter the protein using a 0.2 µm filter. The concentration of the protein solution to be conjugated should be confirmed prior to beginning the conjugation reaction.Note: Conjugation buffer, 0.2 µm filter, and syringes are included in the MSD GOLD SULFO-TAG NHS-Ester Conjugation Packs.2.Equilibrate the protein to be conjugated with MSD GOLD SULFO-TAG NHS-Ester at the conjugation temperature of 23°C.A temperature range of 20°C to 25°C is acceptable. The equilibration can take between 10-30 minutes depending on thevolume of protein.3.Calculate the amount of MSD GOLD SULFO-TAG NHS-Ester stock solution required for the conjugation reaction using theformula depicted below and on the attached worksheet.EXAMPLE❑500 µL of 2 mg/mL antibody❑12:1 challenge ratio❑MSD GOLD SULFO-TAG stock = 3 nmol/µL1000 x 2 mg/mL x 12 x 500 µL = 80 nmol of MSD GOLD SULFO-TAG reagent required150,000 Da80 nmol of SULFO-TAG reagent = 26.7 µL of SULFO-TAG stock solution required for conjugation reaction3 nmol/µL SULFO-TAG stock solution4.Centrifuge the MSD GOLD SULFO-TAG NHS-Ester vial by pulse spinning for 1 minute or gently tap on a soft surface inorder to collect lyophilized material at the bottom of the vial. Reconstitute MSD GOLD SULFO-TAG NHS-Ester immediately prior to use with cold distilled water. For the 2 µmol and 150 nmol sizes of MSD GOLD SULFO-TAG NHS-Ester, dissolve with 200 µL and 50 µL, respectively to generate stock solutions of 10 and 3 nmol/µL. Gently vortex the vial to ensure complete dissolution of all lyophilized material.Note: Reconstituted MSD GOLD SULFO-TAG NHS-Ester may be kept for up to 10 minutes on ice prior to use.5.Add the calculated volume (from Step 3) of reconstituted MSD GOLD SULFO-TAG NHS-Ester to the protein solution andvortex immediately. Discard any remaining unused MSD GOLD SULFO-TAG NHS-Ester.6.Incubate at 23°C for 2 hours; a temperature range of 20°C to 25°C is acceptable. Shield the reaction from light bycovering the tube with aluminum foil or placing it in a dark area (e.g., a closed drawer). Take care to maintain consistent conjugation conditions between multiple preparation lots to ensure conjugation reproducibility.7.Prepare Zeba Spin D esalting Columns, 40K MWCO, towards the end of the incubation period. Remove the column'sbottom closure and loosen the cap. Do not remove the cap. Place the column in a collection tube to remove the storage buffer and wash the column 3 times with Conjugate Storage Buffer. Each preparation step should be carried out by centrifuging the columns, and their respective collection tubes, in a centrifuge with a swinging bucket rotor at 2–8°C.Refer to Table 5 below for the recommended sample volume, wash buffer volumes, collection tube sizes, and centrifugation times for each preparation step.Note: Reaction volumes larger than the capacity of a Zeba column should be distributed over multiple Zeba columns.8.Apply the conjugation reaction to the Zeba column in a dropwise manner following the recommendations in Table 2. Usinga swinging bucket rotor, centrifuge the columns in clean new collection tubes to purify the MSD GOLD SULFO-TAGconjugated protein. The MSD GOLD SULFO-TAG conjugated protein will be captured in the conical tubes. Retain the conjugated material in the conical tubes, and discard the columns.Note: The unconjugated MSD GOLD SULFO-TAG reagent will appear as yellow in the spin column.Table 5: Specifications for Zeba Spin Desalting Columns, 40K MWCOSize of Column (mL) 0.5 2 5 10 Sample Volume Range (µL) 70–100 200–450 300–1,000 1,000–2,000 Wash Buffer Volume 300 µL 1 mL 2.5 mL 5 mL Sample Volumes to use a stacker1N/A <350 µL <750 µL <1,500 µL Optional Stacker Volume N/A 40 µL 100 µL 200 µL Centrifugation Speed 1,500 x g1,000 x g1,000 x g1,000 x gCentrifugation Time(Min) Storage Solution Removal 1 2 2 2 Wash 1 1 4–8 4–8 4–8 Wash 2 1 2–8 2–8 2–8 Wash 32 3 5–8 5–8 5–8 Sample Recovery33–4 6–8 6–8 6–8addition of the protein to the column.2 If column is not mostly white after the third wash, the column may be spun for an additional 1–3 minutes.3 Additional sample recovery spin time is allowed if needed to ensure maximum recovery of sample. Overdrying the resin at this stage will not harm the protein; therefore, spinning for up to 8 minutes is allowed.9.It is recommended to filter the conjugated protein using a 0.2 µm filter. Filtration may cause some loss of the protein.Please refer to page 7 for the recommended filter units.10.Determine the molar protein concentration of the conjugated protein using a standard colorimetric protein assay such asBCA, Bradford, or Lowry.absorbance reading as MSD GOLD SULFO-TAG will absorb light at this wavelength.Note: Do not use an OD28011.Measure the absorbance of the MSD GOLD SULFO-TAG protein conjugate at 455 nm using a spectrophotometer. Dividethe measured value by the path length in cm, and then divide by the extinction coefficient of the label (15,400 M-1cm-1) to obtain the MSD GOLD SULFO-TAG label concentration in moles per liter. For reference, a formula calculation worksheet page is attached.12.To calculate the MSD GOLD SULFO-TAG label:protein conjugation ratio, divide the MSD GOLD SULFO-TAG labelconcentration value determined in step 11 by the molar protein concentration value determined in step 10.13.Antibody conjugates are usually stable for at least 2 years at 2–8°C at a concentration of 1–2 mg/mL; stability of otherprotein types should be determined. Conjugated proteins may be stored frozen at ≤-20°C or ≤-70°C, as long as the protein is stable to freeze-thaw cycles or stored in single-use aliquots. MSD GOLD SULFO-TAG conjugated proteins may be sensitive to extended exposure to light and should be stored in the dark or in amber or opaque vials. Short-term exposure of conjugates to light when carrying out assays is not a concern. If the protein concentration is low (< 0.1 mg/mL), consider adding a carrier protein such as 0.1% MSD Blocker A.Storage, Handling, and StabilityMSD GOLD SULFO-TAG NHS-Ester is supplied as a dry orange-red lyophilized solid. The vials should be stored at ≤-70°C. The expiration date of the product is indicated on the label. Following reconstitution, any remaining unused material should be discarded.FAQs1)What chemicals interfere with MSD GOLD SULFO-TAG conjugation?Primary amines and strong nucleophiles interfere with MSD GOLD SULFO-TAG NHS-Ester conjugation. Common reagents that can interfere with the amine coupling of NHS chemistry are:a)Trisb)Glycinec)Histidined)Azidee)Imidazolef)Glutathioneg)Ammonium ionsh)Glycerol2)What are typical carrier proteins in antibody solutions?a)BSAb)GelatinAntibodies should be obtained in carrier protein-free formulations for labeling with MSD GOLD SULFO-TAG NHS-Ester.Carrier proteins will interfere with MSD GOLD SULFO-TAG NHS-Ester conjugation and cannot be removed with desalting columns.3)What is the minimum amount of material that can be conjugated?Generally, 50-100 µg can be conjugated in PBS (without interfering buffer components) if the protein concentration is high enough (1–2 mg/mL). Otherwise, microconcentrators may be used to concentrate the antibody solution following equilibration of the microconcentrator with PBS.4)Are there alternatives to using Thermo Scientific Zeba Spin D esalting Columns for purifying the MSD GOLDSULFO-TAG conjugated antibody after conjugation?a)Users may purchase commercially available G-50 SEPHADEX columns or prepare G-50 SEPHADEX columns atthe bench. However, some G-50 columns may not be efficient in complete removal of unconjugated material. TheSEPHADEX grade is important. MSD recommends using fine grade SEPHADEX for preparing self-packed gelfiltration columns. Medium Grade SEPHADEX does not provide suitable separations and Superfine SEPHADEXdoes not allow an adequate flow rate without use of a pump. It is not recommended to use PD10 columns or G-25 SEPHADEX spin columns for purification of MSD GOLD SULFO-TAG-conjugated protein as these are notable to separate free MSD GOLD SULFO-TAG reagent from labeled conjugates.b)Alternatively, CENTRICON concentrators or similar microconcentrator products with adequate MWCO (forconcentrator information please refer to page 7) can be used to remove unbound label. Resuspend theconjugation mixture in a larger volume of PBS-0.05% azide, concentrate to a smaller volume, and then repeat theprocess as per the product instructions for desalting applications.c)Post-conjugation purification of proteins with MW < 40,000 Da will require alternative procedures (such as high-resolution size exclusion chromatography, HPLC, FPLC, etc.) because ZEBA Spin Desalting Columns or G-50columns will not provide adequate separation in this size range.5)What is the molecular weight of MSD GOLD SULFO-TAG?Unreacted MSD GOLD SULFO-TAG NHS-Ester has a molecular weight of 1,141 g/mol. After the conjugation reaction, each conjugated MSD GOLD SULFO-TAG adds 1,027 g/mol to the protein.6)What types of material can be conjugated?MSD GOLD SULFO-TAG NHS-Ester is reactive with primary amines. Proteins and large peptides are easily labeled. Fab fragments have also been conjugated successfully.MSD Conjugation Services may be used to conjugate small molecules and peptides. Please contact MSD Scientific Support (Phone: 1-240-314-2798, Email: ScientificSupport@) or your local MSD Application Scientist for details.7)What conjugation ratio is recommended for an IgM?A challenge ratio range from 8:1 to 12:1 may be used for conjugating IgM antibodies. The molecular weight of an IgM is inthe order of 900,000 Da. IgMs can be unstable at higher pHs, therefore conjugation at pH 7.0 to 7.2 may be better than the standard labeling buffer of pH 7.9 used for IgG.8)Are there alternatives to using Conjugation Buffer for the conjugation reaction?For best results, it is recommended to use Conjugation Buffer. However, other buffers can be used for the conjugation reaction provided they are free of amine-containing molecules (i.e., no Tris- or glycine-containing buffers) and preservatives. Affinity-purified antibodies are commonly eluted with high molarity glycine solutions; therefore, it is important that they are properly desalted prior to conjugation. Using alternative conjugation buffers may result in lower incorporation efficiencies.9)What should I do if my application requires the conjugated protein to be in a different buffer?The desalting columns may be equilibrated in a buffer other than PBS if the end application requires storage of the conjugated protein in a non-PBS buffer.10)Will my antibody retain activity after labeling?MSD GOLD SULFO-TAG is a small hydrophilic molecule and generally does not affect the function of its conjugation partner, especially when labeling large proteins such as antibodies. With small molecule or peptide labeling, the addition of MSD GOLD SULFO-TAG may have an effect on binding affinities.11)What is the stability of MSD GOLD SULFO-TAG NHS-Ester?The shelf life of MSD GOLD SULFO-TAG NHS-Ester is 3 years at ≤-70°C. The reagent can be stored for up to 2 years at ≤-10°C with minimal loss of activity. Reagent stability is lower at room temperature or at 2–8°C. At room temperature, there may be a 1/3 to 1/2 loss of active material in a month. Once the reagent is reconstituted, it should be used as soon as possible since the NHS-ester hydrolyzes in water. After reconstitution, the solution may be kept up to 10 minutes on ice with minimal loss of activity.12)What if the protein to be conjugated does not have any primary amine groups?Alternative linking chemistry options are available from MSD which allow non-amine containing molecules to be successfully labeled. These include Thiol-Reactive linker (SULFO-TAG Iodoacetamide), Carboxyl (-COOH) Reactive linker (SULFO-TAG Amine) and Carbohydrate Reactive SULFO-TAG. Please contact MSD Scientific Support (Phone: 1-240-314-2798, Email: ScientificSupport@) or your local MSD Application Scientist for details.13)I have an IgG purified antibody from my protein production group which has been eluted into PBS. Should Idesalt before conjugation?Yes. Tris-glycine is a major component of antibody elution buffers used in the purification procedure. On many occasions,a single desalting into PBS is insufficient. It is recommended to repeat the desalting step into PBS to remove any tracequantities of Tris-glycine that can hinder conjugation with MSD GOLD SULFO-TAG.14)How do I conjugate a high concentration protein solution with MSD GOLD SULFO-TAG?The conjugation reaction will be more efficient at high protein concentrations. It is recommended to use a lower challenge ratio for conjugation to compensate for the increased efficiency.15)How do I conjugate a low concentration protein solution with MSD GOLD SULFO-TAG?MSD recommends the protein concentration to be at least 0.5 mg/mL. If concentrating the protein solution is not feasible, conjugation can be done at a lower concentration, which may result in lower conjugation efficiency. Therefore, the conjugation reaction should be performed at a challenge ratio of 20:1 or higher.16)How do I conjugate small proteins with MSD GOLD SULFO-TAG?Proteins with MW < 40,000 Da can be conjugated by the same chemistry as antibodies; lower challenge ratios may be required for MSD GOLD SULFO-TAG conjugation of small proteins and peptides than for IgGs. The NHS-Ester will react with primary amines such as lysine residues and the N-terminus of proteins and peptides. If there is no primary amine available, a different chemistry will be necessary. Post-conjugation purification of small proteins will require alternative procedures (such as HPLC, FPLC, etc.) because small proteins are not resolved by G-50 columns.17)Why can't I use a spectrophotometer at 280 nm to determine conjugated protein concentration?MSD GOLD SULFO-TAG strongly absorbs at 280 nm and will interfere with any measurement of protein concentration at this wavelength.18)What is the stability of MSD GOLD SULFO-TAG conjugated proteins?MSD GOLD SULFO-TAG conjugated protein is generally as stable as the unconjugated protein if it is stored in the appropriate buffer, concentration, and storage temperature. The conjugated protein should be stored in the dark, either at 2–8°C or frozen in aliquots. Azide should be added for long term storage at 2–8°C to prevent any microbial growth. If the protein concentration is low, consider adding a carrier protein, such as 0.1% MSD Blocker A.19)My antibody did not conjugate very well. What are the possible reasons?The presence of preservatives, carrier protein or residual Tris-glycine or other interfering substances in the conjugation buffer (see FAQ 1 and 2) can reduce conjugation efficiency of the protein. Very low concentrations of the starting material (below 0.5 mg/mL) may also reduce conjugation ratios. It has also been observed that some IgGs label more efficiently than others.20)What components can be removed by buffer exchange or dialysis?Salt, azide, glycerol, buffering agent (e.g., Tris), carbohydrates (e.g., trehalose), and amino acids (e.g., histidine, glycine) can be successfully removed by buffer exchange method.21)Who should I contact if I have any questions on MSD GOLD SULFO-TAG conjugation?Please contact MSD Scientific Support (Phone: 1-240-314-2798, Email: ScientificSupport@) or your local MSD Application Scientist for details.WorksheetDate:MaterialsProtein to be conjugatedConcentration: Vendor:Catalog number: Lot number:Sample PreparationMethod: Buffer:Lot number: Date:Columns/Concentrators: Lot number:MSD GOLD SULFO-TAG NHS-Ester ReconstitutionSize: Lot number:Distilled water:Lot number: Date:Volume of water added to vial: Stock concentration (nmol/µL):Separation and CalculationsBuffer:Lot number: Date:Columns: Lot number:Protein assay kit:Type: Lot number:Pre-Conjugation Calculations1,000 x Protein conc. (mg/mL) x Challenge ratio x Volume of protein solution (µL) = nmol of SULFO-TAG reagent required Protein MW (Da)nmol of SULFO-TAG reagent required = µL of SULFO-TAG stock solution required for conjugation reaction Conc. of SULFO-TAG stock solution (nmol/µL)。
DescriptionLORD Chemlok ® 253H adhesive is a high solids, one-coat adhesive designed to bond compounds based on natural rubber (NR), polyisoprene (IR), styrene-butadiene (SBR), polybutadiene (BR), polychloroprene (CR),nitrile (NBR), butyl (IIR) and EPDM polymers to metals. These metals include carbon and alloy steels, stainless steel, aluminum, magnesium, zinc, copper and copper alloys. It is composed of a mixture of polymers, organic compounds and mineral fi llers dissolved or dispersed in an organic solvent system.Features and Benefi tsConvenient – requires only a single coat for most appli-cations, reducing labor, solvent usage, inventory and shipping costs.Non-Chlorinated Solvent System – suitable for solvent incineration, saving cost of recovery equipment.Environmentally Resistant – provides superior resis-tance to heat, oils and salt spray.ApplicationSurface Preparation – Thoroughly clean metal surfaces prior to adhesive application. Remove protective oils,Chemlok ®253H Adhesivecutting oils and greases by solvent degreasing oralkaline cleaning. Remove rust, scale or oxide coatings by suitable chemical or mechanical cleaning methods.•Chemical CleaningChemical treatments are readily adapted to auto-mated metal treatment and adhesive application lines. Chemical treatments are also used on metal parts that would be distorted by blast cleaning or where tight tolerances must be maintained. Phos-phatizing is a commonly used chemical treatment for steel, while conversion coatings are commonly used for aluminum.•Mechanical CleaningGrit blasting is the most widely used method ofmechanical cleaning. However machining, grinding or wire brushing can be used. Use steel grit to blast clean steel, cast iron and other ferrous metals. Use aluminum oxide, sand or other nonferrous grit to blast clean stainless steel, aluminum, brass, zinc and other nonferrous metals.For further detailed information on surface preparation of specifi c substrates, refer to Chemlok Adhesives appli-cation guide. Handle clean metal surfaces with clean gloves to avoid contamination with skin oils.Typical Properties*Appearance Black Liquid Viscositycps @ 25°C (77°F)300-2000 Brookfi eld VT Spindle 2, 30 rpmseconds20-70Zahn Cup #4Density kg/m 3 982.6-1030.5 (lb/gal)(8.2-8.6)Solids Content by Weight, % 25-30Flash Point, °C (°F) 9 (48)SolventsToluene*Data is typical and not to be used for specification purposes.LORD provides valuable expertise in adhesives and coatings, vibration and motion control, and magnetically responsive technologies. Ourpeople work in collaboration with our customers to help them increase the value of their products. Innovative and responsive in an ever-changing marketplace, we are focused on providing solutions for our customers worldwide ... Ask Us How.LORD Corporation World Headquarters 111 Lord DriveCary, NC 27511-7923 USACustomer Support Center (in United States & Canada)+1 877 ASK LORD (275 5673)For a listing of our worldwide locations, visit .©2010 LORD Corporation OD DS3128 (Rev.4 7/10)Mixing – Thoroughly stir Chemlok 253H adhesive beforeuse, and agitate suffi ciently during use to keep dispersed solids uniformly suspended. Mix drums for 8 hours orlonger at 30-60 rpm before using.Applying – Apply Chemlok 253H adhesive by spray, dip or brush methods. Chemlok 253H adhesive is best suited for dip application.When using Chemlok 253H adhesive as a one-coatadhesive, the dry fi lm thickness should be maintained at17.8-27.9 micron (0.7-1.1 mil), particularly if the bonded assembly is likely to be exposed to severe environments. When used as a covercoat over a primer, the dry fi lm thickness of Chemlok 253H adhesive should be 15.2-20.3 micron (0.6-0.8 mil).• SprayingDilute Chemlok 253H adhesive to a Zahn Cup #2viscosity of approximately 21 seconds. Use a ratio of approximately 50 parts adhesive to 50 parts xylene ortoluene. Tip sizes of 1.07-1.4 mm (0.042-0.055 in) are appropriate. Maintain atomization pressures at 0.345-0.379 MPa (50-55 psi).• D ippingUse full strength. As a one-coat adhesive, a single dipapplication of Chemlok 253H adhesive usually resultsin a dry fi lm thickness of approximately 25.4 micron(1.0 mil).• Brushing Apply full strength.Curing – Chemlok 253H adhesive can be used incompression, transfer and injection molding procedures.Ideal bonding conditions involve a minimum amount of time between loading the adhesive coated parts andelastomer vulcanization. However, Chemlok 253H adhe-sive will resist moderate prebake times without affectingbond performance.Cleanup – Use solvents such as xylene and methyl ethyl ketone (MEK) to remove adhesive before heat is applied.Shelf Life/StorageShelf life is six months from date of shipment when stored in a well ventilated area at 21-27°C (70-80°F) in original, unopened container. Do not store or use near heat, sparks or open flame.Cautionary Information Before using this or any LORD product, refer to the Mate-rial Safety Data Sheet (MSDS) and label for safe use and handling instructions.For industrial/commercial use only. Must be applied bytrained personnel only. Not to be used in householdapplications. Not for consumer use.Values stated in this technical data sheet represent typical values as not all tests are run on each lot of material produced. For formalized product specifi cations for specifi c product end uses, contact the Customer Support rmation provided herein is based upon tests believed to be reliable. In as much as LORD Corporation has no control over the manner in which others may use this information, it does not guarantee the results to be obtained. In addition, LORD Corporation does not guarantee the perfor-mance of the product or the results obtained from the use of the product or this information where the product has been repackaged by any third party, including but not limited to any product end-user. Nor does the company make any express or implied warranty of merchantability or fi tness for a particular purpose concerning the effects or results of such use.Chemlok and “Ask Us How” are trademarks of LORD Corporation or one of its subsidiaries.。
Chemlok® 8007 Blue Primer Technical Data SheetChemlok® 8007 Blue water-based primer is designed to bond elastomers to metals and other substrates when used with Chemlok water-based covercoat adhesives, including Chemlok 8560S-1, Chemlok 8560D-1 and Chemlok 8210 adhesives. It is composed of a mixture of dispersed mineral fillers, organic compounds, resins and polymer latices in an aqueous medium. Features and Benefits:Environmentally Preferred – provides reduced VOC emissions, allowing for a safer work environment. Versatile – functions as an effective primer for many materials such as steel, phosphated steel, nylon, aluminum and brass.Environmentally Resistant – provides excellent resistance to water, salt spray, glycol, oil and heat. Excellent Appearance – provides a smooth coating for use by dip or spray applications. The blue color affords good contrast with black covercoats and prepared metals. Easy to Use – easily redispersed within 5 to 15 minutes of stirring; ready to use directly out of the container without dilution.Application:Surface Preparation – Thoroughly clean metal surfaces prior to application. Remove protective oils, cutting oils and greases by solvent degreasing or alkaline cleaning. Remove rust, scale or oxide coatings by suitable chemical or mechanical cleaning methods.For further detailed information on surface preparationof specific substrates, refer to Chemlok Adhesives application guide.Mixing – Thoroughly mix primer before using to disperse any soft settling which may have occurred during storage. Do not shake. To prevent foaming, mechanical mixing should not exceed 30 rpm. The addition of anti-foaming agents is not recommended.In most cases, dilution is not required. Deionized wateris suggested if dilution is necessary. Add water gradually while stirring either by hand or by using another low-shear mixing method.Applying – Preheat substrates to 49-65°C (120-150°F) prior to spray application of primer. This heat and spray method prevents runs and sags and gives a dry coating ready for covercoat application. Use contaminant-freeair for spraying. All spray equipment, including pressure pots, hoses, guns and nozzles, should be stainless steel or plastic.Dry film thickness of Chemlok 8007 Blue primer should be5.1-12.7 micron (0.2-0.5 mil).Chemlok 8007 Blue Primer — Technical Data SheetParker LordEngineered Materials Group 111 LORD DriveCary, NC 27511-7923USAphone +1 877 ASK LORD (275 5673)Values stated in this document represent typical values as not all tests are run on each lot of material produced. For formalized product specifications for specific product end uses, contact the Customer Support Center.Information provided herein is based upon tests believed to be reliable. In as much as Parker Lord has no control over the manner in which others may use this information, it does not guarantee the results to be obtained. In addition, Parker Lord does not guarantee the performance of the product or the results obtained from the use of the product or this information where the product has been repackaged by any third party, including but not limited to any product end-user. Nor does the company make any express or implied warranty of merchantability or fitness for a particular purpose concerning the effects or results of such use.WARNING — USER RESPONSIBILITY . FAILURE OR IMPROPER SELECTION OR IMPROPER USE OF THE PRODUCTS DESCRIBED HEREIN OR RELATED ITEMS CAN CAUSE DEATH, PERSONAL INJURY AND PROPERTY DAMAGE.This document and other information from Parker-Hannifin Corporation, its subsidiaries and authorized distributors provide product or system options for further investigation by users having technical expertise.The user, through its own analysis and testing, is solely responsible for making the final selection of the system and components and assuring that all performance, endurance, maintenance, safety and warning requirements of theapplication are met. The user must analyze all aspects of the application, follow applicable industry standards, and follow the information concerning the product in the current product catalog and in any other materials provided from Parker or its subsidiaries or authorized distributors.To the extent that Parker or its subsidiaries or authorized distributors provide component or system options based upon data or specifications provided by the user, the user is responsible for determining that such data and specifications are suitable and sufficient for all applications and reasonably foreseeable uses of the components or systems.©2022 Parker Hannifin - All Rights ReservedInformation and specifications subject to change without notice and without liability therefor. Trademarks used herein are the property of their respective owners.OD DS4175 11/22 Rev.3Drying/Curing – If no preheat is employed, parts will dry in 30 minutes to one hour at room temperature.When used in combination with Chemlok covercoat adhesives, Chemlok 8007 Blue primer can be used to bond rubber by compression, transfer and injection molding procedures.Cleanup – Use soap and water to remove wet primer. Dried primer is not water-soluble and must be removed with a ketone-type solvent.Shelf Life/Storage:Shelf life is three months from date of shipment when stored by the recipient at 21-27°C (70-80°F) in original, unopened container. Do not freeze product. Storage below 4°C (40°F) may be detrimental to the adhesive’s properties.Cautionary Information:Before using this or any Parker Lord product, refer to the Safety Data Sheet (SDS) and label for safe use and handling instructions.For industrial/commercial use only. Must be applied by trained personnel only. Not to be used in household applications. Not for consumer use.。
