Expression and purification of an anti-Foot-and-mouth disease virus single chain

屠呦呦：青蒿素的发现者，中国的科学之光Tu Youyou, a renowned Chinese scientist, has made significant contributions to the global fight against malaria. She is best known for her groundbreaking discovery of artemisinin, a drug that has saved millions of lives worldwide. Tu's journey to this remarkable achievement began in the 1960s, when malaria was a major public health problem in China.Born in 1930, Tu Youyou grew up in a family with a strong tradition of medicine. Her interest in science and medicine was piqued at a young age, and she went on to study pharmacology at Peking University. After graduating, she joined the China Academy of Chinese Medical Sciences and began her research on traditional Chinese medicine.In the early 1970s, the Chinese government launched a nationwide campaign to find a treatment for malaria. Tu Youyou and her team were assigned to study traditional Chinese medicine for potential anti-malarial agents. They screened hundreds of herbal remedies and eventuallyidentified a plant called Artemisia annua, or sweet wormwood, as a promising source of anti-malarial compounds. The extraction and purification of the activeingredient from Artemisia annua was a challenging task. Tu Youyou and her team spent years experimenting withdifferent extraction methods and refining the purification process. Finally, in 1972, they succeeded in isolating artemisinin, a compound that was highly effective against malaria parasites.Artemisinin and its derivatives have become the mainstay of anti-malarial treatment worldwide. The drug is particularly effective against drug-resistant strains of malaria, making it a crucial tool in the global fight against this deadly disease.Tu Youyou'sdiscovery of artemisinin has not only saved millions of lives but also brought recognition and honor to China's scientific community. Her work has been recognized by numerous international awards, including the Nobel Prize in Medicine in 2015.Tu Youyou's journey is an inspiration to young scientists and researchers. Her dedication to science,perseverance in the face of challenges, and commitment to improving global health have left a lasting impact on the scientific community. She remains a beacon of hope for those who strive to make a difference in the world through science and research.屠呦呦：青蒿素的发现者，中国的科学之光屠呦呦，这位杰出的中国科学家，在全球抗击疟疾的战斗中做出了重大贡献。

pPICZ A, B, and CPichia expression vectors for selection onZeocin™ and purification of recombinant proteins Catalog no. V190-20Rev. Date: 7 July 2010Manual part no. 25-0148MAN00000034User ManualiiTable of ContentsKit Contents and Storage (iv)Accessory Products (v)Introduction (1)Overview (1)Methods (3)Cloning into pPICZ A, B, and C (3)Pichia Transformation (9)Expression in Pichia (13)Purification (15)Appendix (17)Recipes (17)Zeocin™ (19)Map and Features of pPICZ A, B, and C (21)Lithium Chloride Transformation Method (23)Construction of In Vitro Multimers (24)Technical Support (32)Purchaser Notification (33)References (34)iiiKit Contents and StorageContents The following components are included with Catalog no. V190–20. Note that thepPICZ expression vectors are supplied in suspension.Component QuantityCompositionpPICZ A Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0pPICZ B Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0pPICZ C Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0GS115/pPICZ/lacZ Positive1 stab --Control strainShipping/Storage The components included with Catalog no. V190–20 are shipped on wet ice.Upon receipt, store as directed below.For long-term storage of your positive control stab strain, we recommendpreparing a glycerol stock immediately upon receipt and storing at –80°C.Component ShippingStorage pPICZ A Expression Vector Wet ice Store at –20°CpPICZ B Expression Vector Wet ice Store at –20°CpPICZ C Expression Vector Wet ice Store at –20°CGS115/pPICZ/lacZ positive control strain Wet ice Store at 4°CivAccessory ProductsAdditional ProductsThe products listed in this section are intended for use with the pPICZ vectors.For more information, visit our web site at or contactTechnical Support (page 32).Product QuantityCatalogno. X-33 Pichia strain 1 stab C180-00GS115 Pichia strain 1 stab C181-00KM71H Pichia strain 1 stab C182-00SMD1168H Pichia strain 1 stab C184-00pPICZα A, B, and C 20 μg each V195-20pPIC6α A,B, and C 20 μg each V215-20pPIC6 A, B, and C 20 μg each V210-20pPIC6 Starter Kit 1 kit K210-01Original Pichia Expression Kit 1 kit K1710-01EasySelect™Pichia Expression Kit 1 kit K1740-01Pichia EasyComp™ Transformation Kit 1 kit K1730-01Pichia Protocols 1 book G100-01PureLink™ Gel Extraction Kit 50 preps250 prepsK2100–12K2100–25S.N.A.P ™ Gel Purification Kit 25 preps K1999–25PureLink™ Quick Plasmid Miniprep Kit 50 preps250 prepsK2100–10K2100–11PureLink™ HiPure Plasmid Midiprep Kit 25 preps50 prepsK2100–04K2100–13One Shot® TOP10 (chemically competent E. coli) 10 reactions20 reactionsC4040–10C4040–03One Shot® TOP10 Electrocompetent E. Coli 10 reactions20 reactionsC4040-50C4040-52TOP10 Electrocomp™ Kits 20 reactions C664–55Positope™ Control Protein 5 μg R900-50CIAP (Calf Intestinal Alkaline Phosphatase) 1,000 units 18009–019T4 DNA Ligase 100 units500 units15224–01715224–025Zeocin™ 1g5 gR250-01R250-05β-Gal Assay Kit 1 kit K1455-01β-Gal Staining Kit 1 kit K1465-01E-Gel® Agarose Gels E-Gel® Agarose Gels are bufferless, pre-cast agarose gels designed for fast, convenient electrophoresis of DNA samples. E-Gel® agarose gels are available in different agarose percentage and well format for your convenience.For more details on these products, visit our web site at or contact Technical Support (page 32).Continued on next pagevAccessory Products, ContinuedZeocin™Zeocin™ may be obtained from Invitrogen (see above). For your convenience, the drug is prepared in autoclaved, deionized water and available in 1.25 ml aliquotsat a concentration of 100 mg/ml. The stability of Zeocin™ is guaranteed for sixmonths if stored at –20°C.Detection of Fusion Protein A number of antibodies are available from Invitrogen to detect expression ofyour fusion protein from the pPICZ vector. Horseradish peroxidase (HRP)-conjugated antibodies allow one-step detection in Western blots usingcolorimetric or chemiluminescent detection methods. The amount of antibodysupplied is sufficient for 25 Western Blots.Antibody Epitope Catalogno.Anti-myc R950–25 Anti-myc-HRPDetects the 10 amino acid epitopederived from c-myc (Evans et al., 1985):EQKLISEEDLR951–25Anti-His(C-term) R930–25Anti-His(C-term)-HRPDetects the C-terminal polyhistidine(6xHis) tag (requires the free carboxylgroup for detection) (Lindner et al., 1997):HHHHHH-COOHR931–25Purification of Fusion Protein The polyhistidine (6xHis) tag allows purification of the recombinant fusionprotein using metal-chelating resins such as ProBond™. Ordering information forProBond™ resin is provided below.Product QuantityCatalogno. ProBond™ Purification System 6 purifications K850–01ProBond ™ Purification System with Anti-myc-HRP Antibody1 Kit K852–01ProBond ™ Purification System with Anti-His(C-term)-HRP Antibody1 Kit K853–01ProBond™ Nickel-Chelating Resin 50 ml150 mlR801–01R801–15Purification Columns 50 each R640–50viIntroductionOverviewIntroduction pPICZ A, B, and C are 3.3 kb expression vectors used to express recombinantproteins in Pichia pastoris. Recombinant proteins are expressed as fusions to aC-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag.The vector allows high-level, methanol inducible expression of the gene ofinterest in Pichia, and can be used in any Pichia strain including X33, GS115,SMD1168H, and KM71H. pPICZ contains the following elements:•5′ fragment containing the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest (Ellis et al., 1985; Koutz et al., 1989;Tschopp et al., 1987a)•Zeocin™ resistance gene for selection in both E. coli and Pichia (Baron et al.,1992; Drocourt et al., 1990)•C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis)tag for detection and purification of a recombinant fusion protein (if desired)•Three reading frames to facilitate in-frame cloning with the C-terminalpeptideReference Sources The pPICZ A, B, and C expression vectors may be used with the Original Pichia Expression Kit, and are included in the EasySelect™Pichia Expression Kit (see page v for ordering information). Additional general information about recombinant protein expression in Pichia pastoris is provided in the manuals for the Original Pichia Expression Kit and the EasySelect™Pichia Expression Kit. For more information about the Original Pichia Expression Kit, the EasySelect™Pichia Expression Kit, or their manuals, visit our web site at or contact Technical Support (page 32).More detailed information and protocols dealing with Pichia pastoris may also be found in the following general reference:Higgins, D. R., and Cregg, J. M. (1998) Pichia Protocols. In Methods in Molecular Biology, Vol. 103. (J. M. Walker, ed. Humana Press, Totowa, NJ) (see page v for ordering information).Recommended Pichia Host Strain We recommend using the X-33 Pichia strain as the host for expression of recombinant proteins from pPICZ. Other Pichia strains including GS115, KM71H, and SMD1168H are suitable. The X-33 Pichia strain and other strains are available from Invitrogen (see page v for ordering information). The X-33 Pichia strain has the following genotype and phenotype:Genotype: Wild-typePhenotype: Mut+1Overview, ContinuedExperimental Overview The following table describes the basic steps needed to clone and express your gene of interest in pPICZ.Step Action1 Propagate pPICZ A, B, and C by transformation into a rec A, end A1E. coli strain such as TOP10, DH5 , or JM109.2 Develop a cloning strategy and ligate your gene into one of the pPICZvectors in frame with the C-terminal tag.3 TransformintoE. coli and select transformants on Low Salt LB platescontaining 25 μg/ml Zeocin™.4 Analyze 10–20 transformants by restriction mapping or sequencing toconfirm in-frame fusion of your gene with the C-terminal tag.5 Purify and linearize the recombinant plasmid for transformation intoPichia pastoris.6 TransformyourPichia strain and plate onto YPDS plates containing the appropriate concentration of Zeocin™.7 Select for Zeocin™-resistant transformants.8 Optimize expression of your gene.9 Purify your fusion protein on metal-chelating resin (i.e. ProBond™).Continued on next page2MethodsCloning into pPICZ A, B, and CIntroduction The pPICZ vector is supplied with the multiple cloning site in three readingframes (A, B, and C) to facilitate cloning your gene of interest in frame with theC-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag.Use the diagrams provided on pages 5–7 to help you design a strategy to cloneyour gene of interest in frame with the C-terminal peptide. Generalconsiderations for cloning and transformation are discussed in this section.General Molecular Biology Techniques For assistance with E. coli transformations, restriction enzyme analysis, DNA biochemistry, and plasmid preparation, refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).E. coli Strain Many E. coli strains are suitable for the propagation of the pPICZ vectorsincluding TOP10, JM109, and DH5 . We recommend that you propagate thepPICZ vectors in E. coli strains that are recombination deficient (rec A) andendonuclease A deficient (end A).For your convenience, TOP10 E. coli are available as chemically competent orelectrocompetent cells from Invitrogen (page v).Transformation Method You may use any method of choice for transformation. Chemical transformation is the most convenient for many researchers. Electroporation is the most efficient and the method of choice for large plasmids.Maintenance of Plasmids The pPICZ vectors contain the Zeocin™ resistance (Sh ble) gene to allow selection of the plasmid using Zeocin™. To propagate and maintain the pPICZ plasmids, we recommend using the following procedure:e 10 ng of your vector to transform a rec A, end A E. coli strain like TOP10,DH5 , JM109, or equivalent (see above).2.Select transformants on Low Salt LB plates containing 25 μg/ml Zeocin™ (seepage 17 for a recipe).3.Prepare a glycerol stock from each transformant containing plasmid forlong-term storage (see page 8).Continued on next page3Cloning into pPICZ A, B, and C, ContinuedGeneral Considerations The following are some general points to consider when using pPICZ to express your gene of interest in Pichia:•The codon usage in Pichia is believed to be similar to Saccharomyces cerevisiae.•Many Saccharomyces genes have proven to be functional in Pichia.•The premature termination of transcripts because of "AT rich regions" has been observed in Pichia and other eukaryotic systems (Henikoff & Cohen, 1984; Irniger et al., 1991; Scorer et al., 1993; Zaret & Sherman, 1984). If you have problems expressing your gene, check for premature termination by northern analysis and check your sequence for AT rich regions. It may be necessary to change the sequence in order to express your gene (Scorer et al., 1993).•The native 5´ end of the AOX1 mRNA is noted in the diagram for each multiple cloning site. This information is needed to calculate the size of the expressed mRNA of the gene of interest if you need to analyze mRNA for any reason.Cloning Considerations For proper initiation of translation, your insert should contain an initiation ATG codon as part of a yeast consensus sequence (Romanos et al., 1992). An example of a yeast consensus sequence is provided below. The ATG initiation codon is shown underlined.(G/A)NNATG GTo express your gene as a recombinant fusion protein, you must clone your gene in frame with the C-terminal peptide containing the c-myc epitope and the polyhistidine tag. The vector is supplied in three reading frames to facilitate cloning. Refer to the diagrams on pages 5–7 to develop a cloning strategy.If you wish to express your protein without the C-terminal peptide, be sure to include a stop codon.Construction of Multimeric Plasmids pPICZ A, B, and C contain unique Bgl II and Bam H I sites to allow construction of plasmids containing multiple copies of your gene. For information on how to construct multimers, refer to pages 24–31.Continued on next page4Multiple CloningSite of pPICZ A Below is the multiple cloning site for pPICZ A. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ A from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGTTCCTCAG TTCAAGTTGG GCACTTACGA GAAGACCGGT CTTGCTAGAT TCTAATCAAG AGGATGTCAG AATGCCATTT GCCTGAGAGA TGCAGGCTTC ATTTTTGATA CTTTTTTATTTGTAACCTAT ATAGTATAGG ATTTTTTTTG TCATTTTGTT 1218Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming site TGA GTTTTAGCCT TAGACATGAC AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT GGGCCC GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´polyadenylation sitePolyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho ISac II Not I Apa I 104210981158871931991Continued on next pageMultiple CloningSite of pPICZ B Below is the multiple cloning site for pPICZ B. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ B from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGA CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC 1216Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming site TGA GTTTGTAGCC TTAGACATGA AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATTATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT TCTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´ polyadenylation site Polyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho ISac II Not I Xba I 104010961156871931991Continued on next pageMultiple CloningSite of pPICZ C Below is the multiple cloning site for pPICZ C. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ C from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGA CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC 1217Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming siteTGA GTTTGTAGCC TTAGACATGA AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT ACGTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´ polyadenylation site Polyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho I Sac II Not I Sna B I 104110971157871931991Continued on next pageE. coli Transformation Transform your ligation mixtures into a competent rec A, end A E. coli strain(e.g. TOP10, DH5, JM109) and select on Low Salt LB agar plates containing25 μg/ml Zeocin™ (see below). Note that there is no blue/white screening for the presence of insert with pPICZ A, B, or C. Once you have obtained Zeocin™-resistant colonies, pick 10 transformants and screen for the presence and orientation of your insert.Important To facilitate selection of Zeocin™-resistant E. coli, the salt concentration of the medium must remain low (<90 mM) and the pH must be 7.5. Prepare Low Salt LB broth and plates using the recipe in the Appendix, page 17.Failure to lower the salt content of your LB medium will result in non-selection due to inhibition of the drug.C We recommend that you sequence your construct to confirm that your gene is in the correct orientation for expression and cloned in frame with the C-terminal peptide (if desired). Refer to the diagrams on pages 5–7 for the sequences and location of the priming sites.Preparing a Glycerol Stock Once you have identified the correct clone, be sure to purify the colony and make a glycerol stock for long-term storage. It is also a good idea to keep a DNA stock of your plasmid at –20°C.1.Streak the original colony out on an Low Salt LB plate containing 25 μg/mlZeocin™. Incubate the plate at 37°C overnight.2.Isolate a single colony and inoculate into 1–2 ml of Low Salt LB containing25 μg/ml Zeocin™.3.Grow the culture to mid-log phase (OD600 = 0.5–0.7).4.Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to acryovial.5.Store at –80°C.Plasmid Preparation Once you have cloned and sequenced your insert, generate enough plasmid DNA to transform Pichia (5–10 μg of each plasmid per transformation). We recommend isolating plasmid DNA using the PureLink™ Quick Plasmid Miniprep Kit or the PureLink™ HiPure Plasmid Midiprep Kit (page v), or CsCl gradient centrifugation.Once you have purified plasmid DNA, proceed to Pichia Transformation, next page.Pichia TransformationIntroduction You should now have your gene cloned into one of the pPICZ vectors. Yourconstruct should be correctly fused to the C-terminal peptide (if desired). Thissection provides general guidelines to prepare plasmid DNA, transform yourPichia strain, and select for Zeocin™-resistant clones.Zeocin™ Selection We generally use 100 μg/ml Zeocin™ to select for transformants when using the X-33 Pichia strain. If you are transforming your pPICZ construct into anotherPichia strain, note that selection conditions may vary. We recommendperforming a dose response curve to determine the appropriate concentration ofZeocin™ to use for selection of transformants in your strain.Method of Transformation We do not recommend spheroplasting for transformation of Pichia with plasmids containing the Zeocin™ resistance marker. Spheroplasting involves removal of the cell wall to allow DNA to enter the cell. Cells must first regenerate the cell wall before they are able to express the Zeocin™ resistance gene. For this reason, plating spheroplasts directly onto selective medium containing Zeocin™ does not yield any transformants.We recommend electroporation for transformation of Pichia with pPICZ A, B, or C. Electroporation yields 103 to 104 transformants per μg of linearized DNA and does not destroy the cell wall of Pichia. If you do not have access to an electroporation device, use the LiCl protocol on page 23 or the Pichia EasyComp™Transformation Kit available from Invitrogen (see below).PichiaEasyComp™Transformation Kit If you wish to perform chemical transformation of your Pichia strain with pPICZ A, B, or C, the Pichia EasyComp™ Transformation Kit is available from Invitrogen (see page v for ordering information). The Pichia EasyComp™ Transformation Kit provides reagents to prepare 6 preparations of competent cells. Each preparation will yield enough competent cells for 20 transformations. Competent cells may be used immediately or frozen and stored for future use. For more information, visit our web site at or contact Technical Support (page 32).Important Since pPICZ does not contain the HIS4 gene, integration can only occur at the AOX1 locus. Vector linearized within the 5´ AOX1 region will integrate by gene insertion into the host 5´ AOX1 region. Therefore, the Pichia host that you use will determine whether the recombinant strain is able to metabolize methanol (Mut+) or not (Mut S). To generate a Mut+ recombinant strain, you must use a Pichia host that contains the native AOX1 gene (e.g. X-33, GS115, SMD1168H). If you wish to generate a Mut S recombinant strain, then use a Pichia host that has a disrupted AOX1 gene (i.e. KM71H).Continued on next pageHis4 Host Strains Host strains containing the his4 allele (e.g. GS115) and transformed with thepPICZ vectors require histidine when grown in minimal media. Add histidine toa final concentration of 0.004% to ensure growth of your transformants.The pPICZ vectors do not contain a yeast origin of replication. Transformantscan only be isolated if recombination occurs between the plasmid and the Pichiagenome.Materials Needed You will need the following items:Note: Inclusion of sorbitol in YPD plates stabilizes electroporated cells as they appear tobe somewhat osmotically sensitive.•5–10 μg pure pPICZ containing your insert•YPD Medium•50 ml conical polypropylene tubes• 1 liter cold (4°C) sterile water (place on ice the day of the experiment)•25 ml cold (4°C) sterile 1 M sorbitol (place on ice the day of the experiment)•30°C incubator•Electroporation device and 0.2 cm cuvettes•YPDS plates containing the appropriate concentration of Zeocin™ (seepage 18 for recipe)Linearizing YourpPICZ ConstructTo promote integration, we recommend that you linearize your pPICZ constructwithin the 5′ AOX1 region. The table below lists unique sites that may be used tolinearize pPICZ prior to transformation. Other restriction sites are possible.Note that for the enzymes listed below, the cleavage site is the same for versionsA, B, and C of pPICZ. Be sure that your insert does not contain the restriction siteyou wish to use to linearize your vector.Enzyme Restriction Site (bp) SupplierSac I 209 ManyPme I 414 New England BiolabsBst X I 707 ManyRestriction Digest 1.Digest ~5–10 μg of plasmid DNA with one of the enzymes listed above.2.Check a small aliquot of your digest by agarose gel electrophoresis forcomplete linearization.3.If the vector is completely linearized, heat inactivate or add EDTA to stopthe reaction, phenol/chloroform extract once, and ethanol precipitate using1/10 volume 3 M sodium acetate and 2.5 volumes of 100% ethanol.4.Centrifuge the solution to pellet the DNA, wash the pellet with 80% ethanol,air-dry, and resuspend in 10 μl sterile, deionized water. Use immediately orstore at –20°C.Continued on next pagePreparation of Pichia for Electroporation Follow the procedure below to prepare your Pichia pastoris strain for electroporation.1. Grow 5 ml of your Pichia pastoris strain in YPD in a 50 ml conical tube at30°C overnight.2. Inoculate 500 ml of fresh medium in a 2 liter flask with 0.1–0.5 ml of theovernight culture. Grow overnight again to an OD600 = 1.3–1.5.3. Centrifuge the cells at 1500 × g for 5 minutes at 4°C. Resuspend the pelletwith 500 ml of ice-cold (0–4°C), sterile water.4. Centrifuge the cells as in Step 3, then resuspend the pellet with 250 ml ofice-cold (0–4°C), sterile water.5. Centrifuge the cells as in Step 3, then resuspend the pellet in 20 ml of ice-cold (0–4°C) 1 M sorbitol.6. Centrifuge the cells as in Step 3, then resuspend the pellet in 1 ml of ice-cold(0–4°C) 1 M sorbitol for a final volume of approximately 1.5 ml. Keep the cells on ice and use that day. Do not store cells.Transformation by Electroporation 1.Mix 80 μl of the cells from Step 6 (above) with 5–10 μg of linearized pPICZDNA (in 5–10 μl sterile water) and transfer them to an ice-cold (0–4°C)0.2 cm electroporation cuvette.2.Incubate the cuvette with the cells on ice for 5 minutes.3.Pulse the cells according to the parameters for yeast (Saccharomycescerevisiae) as suggested by the manufacturer of the specific electroporation device being used.4.Immediately add 1 ml of ice-cold 1 M sorbitol to the cuvette. Transfer thecuvette contents to a sterile 15 ml tube.5.Let the tube incubate at 30°C without shaking for 1 to 2 hours.6.Spread 50-200 μl each on separate, labeled YPDS plates containing theappropriate concentration of Zeocin™.7.Incubate plates for 2–3 days at 30°C until colonies form.8.Pick 10–20 colonies and purify (streak for single colonies) on fresh YPD orYPDS plates containing the appropriate concentration of Zeocin™.Continued on next pageGenerally, several hundred Zeocin™-resistant colonies are generated using theprotocol on the previous page. If more colonies are needed, the protocol may bemodified as described below. Note that you will need ~20, 150 mm plates withYPDS agar containing the appropriate concentration of Zeocin™.1. Set up two transformations per construct and follow Steps 1 through 5 ofthe Transformation by Electroporation protocol, page 11.2. After 1 hour in 1 M sorbitol at 30°C (Step 5, previous page), add 1 ml YPDmedium to each tube.3. Shake (~200 rpm) the cultures at 30°C.4. After 1 hour, take one of the tubes and plate out all of the cells by spreading200 μl on 150 mm plates containing the appropriate concentration ofZeocin™.5. Optional: Continue incubating the other culture for three more hours (for atotal of four hours) and then plate out all of the cells by spreading 200 μl on150 mm plates containing the appropriate concentration of Zeocin™.6. Incubate plates for 2–4 days at 30°C until colonies form.Mut Phenotype If you used a Pichia strain containing a native AOX1 gene (e.g. X-33, GS115,SMD1168H) as the host for your pPICZ construct, your Zeocin™-resistanttransformants will be Mut+. If you used a strain containing a deletion in theAOX1 gene (e.g. KM71H), your transformants will be Mut S.If you wish to verify the Mut phenotype of your Zeocin™-resistant transformants,you may refer to the general guidelines provided in the EasySelect™PichiaExpression Kit manual or the Original Pichia Expression Kit manual or topublished reference sources (Higgins & Cregg, 1998).You are now ready to test your transformants for expression of your gene ofinterest. See Expression in Pichia, next page.。

快速银染试剂盒产品简介：快速银染试剂盒(Fast Silver Stain Kit)是一种快速简单、可用于SDS-PAGE或非变性PAGE等蛋白银染的试剂盒。

本试剂盒也可以用于2D凝胶的银染，并且染色后和后续的质谱检测兼容。

本试剂盒只需一小时左右即可观察到蛋白条带，90分钟内可以完成2块凝胶的银染。

对于BSA蛋白，检测灵敏度可以达到0.3ng蛋白。

无需使用有毒的甲醇。

本试剂盒可足够用于25块常规的8×10cm凝胶的银染。

保存条件：室温保存，一年有效。

银溶液(100X)和银染显色加速液(2000X)需避光保存。

注意事项：由于银染非常灵敏，操作时请注意尽量使用高纯度的水，并确保所使用的器皿非常清洁，最好使用洁净的玻璃器皿。

操作时必须戴手套，避免皮肤和凝胶直接接触。

需自备乙醇、乙酸及MilliQ级纯水或双蒸水。

下述使用说明中各种溶液的使用量适用于大小为8×10cm厚度为0.75-1mm的凝胶。

对于更大的凝胶，各种溶液的使用量需按凝胶面积的比例放大，对于更厚的凝胶，作用时间需按照厚度的比例适当延长。

本说明书所指的室温为20-25℃，操作温度较低时由于溶液的扩散能力下降，各步骤需适当延长时间。

银染基本显色液(5X)在低温环境下可能会出现少量沉淀，可在30-50℃水浴中溶解，并充分混匀后使用。

至少后续稀释至1X后须确保完全溶解。

为了您的安全和健康，请穿实验服并戴一次性手套操作。

使用说明：1.固定：电泳结束后，取凝胶放入约100ml固定液中，在摇床上室温摇动20分钟，摇动速度为60-70rpm。

固定40分钟以上甚至过夜可以进一步降低背景。

固定液的配制：依次加入50ml乙醇、10ml乙酸和40ml MilliQ级纯水或双蒸水，混匀后即成100ml固定液。

2.30%乙醇洗涤：弃固定液，加入100ml 30%乙醇，在摇床上室温摇动10分钟，摇动速度为60-70rpm。

30%乙醇的配制：70ml MilliQ级纯水或双蒸水中加入30ml乙醇，混匀后即成100ml 30%乙醇。

化学论文英文版Jun-Ke Wang, Ying-Xiao Zong, Xi-Cun Wang, Yu-Lai Hu, Guo-Ren Yue.Synthesisof N-benzothiazol-2-yl-amides by Pd-catalyzed C(sp2)-H functionalization[J]. CCL, 2015,26(11): 1376-1380Synthesis of N-benzothiazol-2-yl-amides byPd-catalyzed C(sp2)-H functionalizationJun-Ke Wang a,b,c, Ying-Xiao Zong a,b, Xi-Cun Wang a,b, Yu-Lai Hu a,b,Guo-Ren Yue aa Key Laboratory of Hexi Corridor Resources Utilization of Gansu Universities, College of Chemistry and Chemical Engineering, Hexi University, Zhangye 734000, China;b Gansu Key Laboratory of Polymer Materials, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070, China;c Gansu Engineering Laboratory of Applied Mycology, Hexi University, Zhangye 734000, ChinaReceived 11 May 2015, Received in revised form 29 June 2015, Accepted 1 July 2015,Available online 10 August 2015.E-mail addresses: wangxicun@;huyulai@Abstract: A catalytic synthesis of N-benzothiazol-2-yl-amides from1-acyl-3-(phenyl)thioureas was achieved in the presence of a palladium catalyst through the C(sp2)-H functionalization/C-S bond formation. This synthetic methodology can produce various N-benzothiazol-2-yl-amides in high yields with good functional group tolerance.Key words: Benzothiazole Pd-catalyzed1-Acyl-3-phenylthiourea C-H functionalization L-Proline1. IntroductionThe benzothiazole moiety is an important scaffold due to its widespread occurrence in bioactive natural products,pharmaceuticals, organic optoelectronic materials,and ligands for phosphorescent complexes [1-4]. In particular,substituted Nbenzothiazol- 2-yl-amides are an important class of heterocyclic compounds that exhibit a wide range of biological properties [5-9] such as ubiquitin ligaseinhibition [5],antitumor [6],antirotavirus infections [7],modulating the adenosine receptor [8, 9],and the nuclear hormone receptor [9]. For example,the N-benzothiazol-2- yl-cyclohexanecarboxamide,as a new anticancer drug,was selected as one of the most promising screening hit compounds (Fig. 1) [6]. The acylation reaction from2-aminobenzothiazole,one of the classical methods for the preparation of these molecules [5, 6],is known for the limited diversity of the commercially available starting materials. Furthermore,the preparation of 2-aminobenzothiazole also required the use of the toxic bromine.The past several years have witnessed the great progress in the development of the C-S bond formation promoted by transition metals,which can provide moreefficient,practical,and straightforward approaches to valuable sulfur-containing compounds [10, 11]. However,these methods have been mainly focused on the‘‘traditional’’ cross-coupling reactions of ArX (X = Cl,Br,I,OTf,and B(OH)2) and sulfides [12-39]. To achieve greener and more atomeconomic C-S bond formations,transition metal-catalyzed direct oxidative cross-coupling of C-H bonds and sulfides would be ideal [40-47].In our previous work,we have shown that N-benzothiazol-2-ylamides can be synthesized smoothly by Cu-catalyzed intramolecular cyclization of various substituted 1-acyl-3-(2-bromophenyl) thioureas [48]. This method can provide more diversiform Nbenzothiazol- 2-yl-amides through the carbon-heteroatom formation under relatively mild conditions and avoid the use of the toxic bromine. However,the drawback of this procedure is the limited diversity of the commercially available starting materials due to the use of substituted ortho-haloarylamines. In order to further extend the diversity of N-benzothiazol-2-yl-amides,we have recently demonstrated an efficient intramolecular cyclization of substituted 1-acetyl-3-(2-phenyl)thiourea catalyzed by iron through C-H functionalization [49]. This method can provide more diversiformN-benzothiazol-2-yl-amides under relatively mild conditions. However,the purification of the target compounds is challenging using the column chromatography or recrystallization,since it is inescapable to obtain 1-acetyl-3-phenylurea whose polarity is similar to that of 1-acetyl-3-(2-phenyl)thiourea. Recently,Doi’s group[46] reported a Pd-catalyzed synthesis of 2-substituted benzothiazoles via a C-H Functionalization reaction. Therefore,we envisioned that Pd-catalyzed cyclization of 1-acyl-3-(2-phenyl)- thiourea 1would represent a viable method for the formation and purification of substituted N-benzothiazol-2-yl-amides 2(Scheme 1).2. ExperimentalAll reagents were commercially available and used as supplied. Dimethyl sulfoxide (DMSO) was dried and distilled from calcium hydride. N,N-Dimethylformamide (DMF),toluene,DME and CH3CN were dried prior to use using standard methods. Unless otherwise stated,analytical grade solvents and commercially available reagents were used as received. Thin layer chromatography (TLC) employed glass 0.20 mm silica gel plates. Flash chromatography columns were packed with 200-300 mesh silica gel.All new compounds were characterized by IR,1H NMR,13C NMR and HRMS. The known compounds were characterized by 1H NMR, 13C NMR and HRMS. The IR spectra were run on a Nicolete spectrometer (KBr). The 1H NMR and 13C NMR spectra were recorded on a BRUKER AVANCEIII 400 MHz spectrometer. The chemical shifts (d) were given in parts per million relative to an internal standard tetramethylsilane. High resolution mass spectra (HRMS) were measured with a Waters Micromass GCT instrument and accurate masses were reported for the molecular ion (M+). Melting points were determined on a Perkin-Elmer differential scanning calorimeter and the thermometer was uncorrected.2.1. General procedure for the synthesis of1-acyl-3-arylthioureas [49, 50]To a 25 mL round-bottom flask equipped with a magnetic stirring bar was added acyl chloride (10 mmol),NH4SCN (15 mmol) and CH2Cl2 (20 mL),followed by PEG-400 (0.1 mmol). The mixture was stirred for approximately 3 h at room temperature. Aromatic amine (10 mmol) was added to the mixture and stirred for another 2 h at room temperature. The solvent was removed under reduced pressure to give the resulting residue as a solid,which was washed with water three times,to give the crude product.The analytical samples were obtained by recrystallization from C2H5OH in good yields ([4TD$DIF]88%-98%).2.2. General procedure for the synthesis ofN-benzothiazol-2-ylamides by aPd-catalysed C(sp2)-H functionalization reactionA round-bottom flask equipped with a stirring bar was charged with1-acyl-3-arylthioureas (1 mmol),PdCl2 (10 mol%),CuI (20 mol%),Cs2CO3 (2 equiv.),and L-proline (20 mol%) in 5 mL of DMSO. The mixture was stirred at 100 ℃for the indicated time in Table 2. After cooling to room temperature,the reaction mixture was extracted with ethyl acetate (10 mL × 3). The organic layers were combined,dried over Na2SO4 and concentrated under reduced pressure,and then purified by silica gel chromatography (acetone/petroleum ether = 1:4) to yield the desired product2.N-(4-Ethylbenzo[d]thiazol-2-yl)acetamide (2f): A gray solid (80% yield); mp:264-268 ℃; IR (cm-1): 3169.9,2990.1,2359.9, 1661.1,1550.4; 1H NMR (400MHz,CDCl3): δ 9.42 (s,1H),7.67 (dd, 1H,J = 6.3,2.9 Hz),7.27 (dd,2H,J = 4.4,1.9 Hz),3.04 (q,2H, J = 7.6 Hz),2.28 (s,3H),1.34 (t,3H,J = 7.6 Hz); 13C NMR (100 MHz,CDCl3): δ171.64(s),156.91 (s),146.45 (s),136.81 (s),131.98 (s), 125.25 (s),124.22 (s),118.92 (s),25.36 (s),23.51 (s),14.79 (s); HRMS calcd. for C11H12N2OS [M]+:220.0670; found [5TD$DIF]200.0678.N-(6-Fluorobenzo[d]thiazol-2-yl)acetamide (2 g): A white solid (94% yield); mp:224-231 ℃; IR (cm-1): 3207.8,3071.0,2983.9, 2360.4,1689.2; 1H NMR (400MHz,CDCl3): δ 7.70 (dd,1H,J = 8.9, 4.6 Hz),7.53 (dd,1H,J = 8.0,2.5 Hz),7.19 (td,1H,J = 8.9,2.6 Hz), 2.31 (s,3H); 13C NMR (100 MHz,CDCl3): δ 168.33 (s),160.93 (s), 158.50 (s),121.30 (d,J = 9.1 Hz),114.75 (s),108.09 (s),107.82 (s), 23.46 (s); HRMS calcd. for C9H7FN2OS [M]+: 210.0263; found 210.0256.3. Results and discussionWhile not commercially available,benzothioureas are stable and easilysynthesized [50, 51] from inexpensive starting materials in high yields on a multigram scale. Following Scheme 2,the synthesis of benzothioureas can be achieved in a straightforward manner starting from inexpensive aryl acid chloride and arylamines. Aryl acid chloride was treated with ammonium sulfocyanide in the presence of PEG-400in CH2Cl2,followed by the addition of arylamines,to obtain 1-arylacyl-3-phenylthiourea in good to excellent yields. This intermediate can be used directly without further purifications.In a preliminary experiment,we investigated the intramolecular C-S bond formation of 1-acetyl-3-phenylthiourea utilizing PdCl2 (20%) and a mild base (K2CO3,2 equiv.) in DMSO for 20 h at 100 ℃(Table 1,entry 1). However,the reaction almost failed to take place. Subsequently,we screened several metal salts as cocatalysts, includingAlCl3,CuCl2,Cu(OAc)2,CoCl2,NiCl2,FeCl3,CuI, and CuCl,and found that the addition of CuI considerably enhanced this reaction (Table 1,entries 2-8). However,the desired yield was still not obtained. Surprisingly,when Doi’s condition was used,the yield was still very low (42%) (Table 1,entry 9). Generally,the choice of the ligands is important for the reaction catalyzed by the metal,which prompted us to explore the effect of several bidentate ligands. We carried out the reaction of 1-acetyl-3-phenylthiourea by screening these ligands,such as 1,10-phenanthroline,β-keto esters,β-diketones,andL-proline. (Table 1,entries 10-13),and we were pleased to find that the use of these ligands can notably improve the yield of the product under the same conditions,and that L-proline proved to be the best among an array of ligands tested (Table 1,entry 14). When the amount of CuI and PdCl2 was decreased to 20 mol% and 10mol%,respectively,the catalytic activity was maintained (Table 1,entry 14). Furthermore,we also investigated other bases (Cs2CO3 and K3PO4) (Table 1,entries 15- 16),solvents (DMF,DME,and toluene) (Table 1,entries 17-19) and reaction time (Table 1,entries 20-21). When only CuI was used in this cyclization,no reaction can take place (Table 1,entry 22). Thus, the optimized reaction conditions are as the follows: substrate (1 mmol),PdCl2 (10 mol%),CuI (20 mol%),Cs2CO3 (2 equiv.), L-proline (20 mol%) in DMSO (4 mL) within 8 h at 100 ℃.In response to this encouraging result,we used a range of substituted1-acetyl-3-(phenyl)thioureas to investigate the scope and limitation of this reaction. The corresponding products were obtained in excellent yields (88%-98%). The results obtained under the optimized conditions are listed in Table 2. Initially,the substituents of phenyl were screened. The results demonstrate that little effect of the substituted groups on the benzene ring was observed for this transformation.Furthermore,substituents at different positions of the phenyl ring do not significantly affect the efficiency (Table 2,entries 1-8). It is noteworthy that the halosubstituted benzenes survived leading to halo-substituted products,which can be used for further transformations (Table 2, entries 2,7,8 and 11). In order to make the new Sankyo investigational drugs,the R group was selected as a cyclohexyl to give the corresponding products (Table 2,entries 10-12).Although extensive studies on reaction mechanism have not yet been carried out,the proposed mechanism can be proposed according to the similar palladium-catalyzed processes [51] (Scheme 3). 1-Acetyl-3-(phenyl)thiourea was converted to the thioenolate in the presence of Cs2CO3. Pre-association of the sulphur atom in the thioenolate to Pd(OAc)2 facilitates the orthopalladation process with the concomitant release of chloride ion. The formation of the six-membered palladacycle and the subsequent reductive elimination leads to N-benzothiazol-2-yl-amide and Pd(0). The Pd(0) species are reoxidized to Pd(II) by CuI,thus completing the catalytic cycle.4. ConclusionIn conclusion,we have achieved an efficient intramolecular cyclization of substituted 1-acetyl-3-(2-phenyl) thioureas catalyzed by palladium(II) catalysts through C(sp2)-H functionalization. This method can provide more diversiform N-benzothiazol-2-yl-amides efficiently and quickly in high yields under relatively mild conditions. The combination of the generality with respect to the substrate scope and facile accessibility to the starting materials may generate numerous synthetic possibilities. 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Organic Chemistry Lab ⅠOrganic Chemistry Lab Ⅰ is a course that introduces students to the basic principles and techniques of organic chemistry. In this lab, students get a hands-on experience with the chemical reactions, properties, and identification of organic compounds. The lab is designed to teach students how to carry out various organic chemistry experiments using different laboratory equipment.The first experiment in the Organic Chemistry Lab Ⅰ course is the extraction and purification of caffeine from tea leaves. In this experiment, students extract caffeine from tea leaves using a Soxhlet extractor and purify it using recrystallization. The experiment is intended to teach students the concept of solubility and its application in separating organic compounds, as well as the importance of purity in organic synthesis.The second experiment is the synthesis of esters, where students ethylate benzoic acid with ethanol using sulfuric acid as a catalyst. 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This experiment teaches students the importance of functional groups in organic chemistry and their identification using chemical tests.The fifth experiment is the synthesis of aspirin. In this experiment, students synthesize aspirin from salicylic acid and acetic anhydride. The product is then purified using recrystallization. This experiment teaches students the technique of acetylation and the importance of purification in organic synthesis.The Organic Chemistry Lab Ⅰ course also includes teaching of different laboratory techniques such as distillation, reflux, titration, and chromatography. The students are also taught laboratory safety measures to ensure their safety while working in the lab.One of the significant benefits of the Organic Chemistry Lab Ⅰcourse is that it prepares students for more advanced courses in organic chemistry. The hands-on experience with various organic chemistry experiments enhances the students’ und erstanding of the concepts and principles of organic chemistry, which they can apply in chemical research and development.In conclusion, the Organic Chemistry Lab Ⅰ course is an important course for students interested in organic chemistry. The course provides students with hands-on experience with various organic chemistry experiments, which enables them to understandthe principles and techniques applied in organic synthesis. The course also initiates the students into basic laboratory techniques and safety measures. Overall, the Organic Chemistry Lab Ⅰcourse is an essential foundation for students who wish to pursue a career in organic chemistry or related fields.The field of organic chemistry is vast and ever-expanding. It is a discipline that deals with the study of carbon-based compounds and their properties, reactions, and applications. Organic chemistry finds its applications in several fields, including pharmaceuticals, agrochemicals, biotechnology, and material science. Therefore, a course like Organic Chemistry Lab Ⅰ is necessary for students to understand the foundational principles and techniques in this field.One of the most critical aspects of the Organic Chemistry Lab Ⅰcourse is the hands-on experience in the laboratory. The laboratory experiments provide students with the opportunity to apply theoretical knowledge to practical applications. For instance, in the caffeine extraction experiment, students learn about the principle of solubility and its application in separating organic compounds. The experiment also teaches how to purify the extracted compound using recrystallization. Such experiments enhance the students’ understanding of organic synthesis techniques, which they can later apply in research and development of organic compounds.The synthesis of esters is another experiment in the Organic Chemistry Lab Ⅰ course that has practical applications. Esters are used in various industries, including the food and beverage industry, as fragrances and flavors. In this experiment, students learn the technique of ethylation and the importance of a catalyst in organic synthesis. They also learn to apply fractional distillationfor separation and purification of the synthesized esters.The Organic Chemistry Lab Ⅰ course also teaches the preparation and properties of Grignard reagents, which are essential in organic synthesis. Grignard reagents have a wide range of applications in the pharmaceutical industry, such as the synthesis of anti-cancer drugs. By learning the principle of organometallic chemistry and the use of Grignard reagents, students develop an understanding of the reaction mechanisms involved in organic synthesis.The identification of functional groups experiment teaches students how to use different chemical tests to determine the functional groups present in organic compounds. The knowledge of the functional groups is vital in organic chemistry, as it determines the properties and reactivity of the compounds. The tests, such as the Tollens test and the Fehling’s test, give stud ents practical applications of the different organic chemistry concepts learned in the classroom.The synthesis of aspirin experiment teaches students how to use acetic anhydride to synthesize aspirin from salicylic acid. Aspirin is one of the most widely used non-steroidal anti-inflammatory drugs (NSAIDs) and has several applications in medicine. This experiment teaches the students the principles of acetylation and the importance of purification in organic synthesis. The students also learn how to perform recrystallization, a technique used to purify organic compounds in the laboratory.Along with the experimental techniques, Organic Chemistry Lab Ⅰ course also teaches various laboratory techniques such asdistillation, reflux, titration, and chromatography. These techniques are essential for organic synthesis and can be used in various industries, including the pharmaceutical and chemical industries.Safety is also a significant concern in any laboratory, and the Organic Chemistry Lab Ⅰ course teaches students to follow safety procedures while working in the laboratory. The course provides guidelines on handling chemicals, using laboratory equipment, and disposing of waste.The Organic Chemistry Lab Ⅰ course is an important foundation for students interested in a career in organic chemistry or related fields. Organic chemistry is a rapidly-growing field, and there is a high demand for professionals with experience in the principles and techniques of organic synthesis. The foundational knowledge and practical experience gained from the course provide students with the necessary skills to pursue higher studies in organic chemistry or to undertake research and development in the field. The benefits of the Organic Chemistry Lab Ⅰ course extend beyond the field of organic chemistry. The course also teaches problem-solving, critical thinking, and analytical skills that are transferable to other fields. These skills include interpreting data, identifying patterns, and making logical conclusions, which are essential in various industries.In conclusion, the Organic Chemistry Lab Ⅰ course provides students with the foundational principles and techniques of organic chemistry. The course teaches students the practical applications of organic chemistry concepts through laboratory experiments such asthe extraction and purification of caffeine, synthesis of esters, preparation of Grignard reagents, identification of functional groups, and synthesis of aspirin. The course also provides students with an understanding of laboratory techniques and safety procedures. Overall, the Organic Chemistry Lab Ⅰ course is an integral course for students who wish to pursue a career in organic chemistry or related fields.。

Data SheetANTI-FLAG® M1 Agarose Affinity Gel A4596Product DescriptionThe FLAG® peptide sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK), is one of the most widely used protein tags in recombinant protein expression and purification.1-3 The ANTI-FLAG® M1 Agarose Affinity Gel is a covalent conjugate of a purified mouse IgG2B monoclonal antibody to agarose by a hydrazide linkage. The ANTI-FLAG® M1 antibody has a binding specificity for FLAG®-tagged proteins with the FLAG®-tag at their N-terminus. This product is useful for calcium-mediated purification of FLAG®fusion proteins.The binding specificity is at the free N-terminus of the FLAG® sequence (N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C). Several theses4-5 and dissertations7-26 cite use of this product in their research protocols. ReagentThe ANTI-FLAG® M1 Affinity Gel is supplied as a suspension in 50% glycerol containing 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4, and0.02% (w/v) sodium azide.Storage/StabilityStore the resin as supplied at –20 °C. Store columns of ANTI-FLAG® Affinity Gel as indicated in the following procedure. Do not store the gel at freezing temperatures in the absence of glycerol.Precautions and DisclaimerFor R&D use only. Not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.ProcedurePurification of FLAG® Fusion Proteins withANTI-FLAG® M1 Affinity Gel•Pre-equilibrate the column and all buffers.Perform all steps at room temperature.•If there is a problem with proteases, perform column chromatography at 2-8 °C. •Cellular debris and particulate matter can clog the column and must be removed prior to purification. •Highly viscous samples containing chromosomalDNA or RNA can also clog the column. Thesesamples should be sonicated or treated withnuclease to reduce the viscosity.•Amino-terminal-FLAG-BAP™ positive control proteins can be used to verify the functionality of the gel.•The ANTI-FLAG® M1 Affinity Gel is resistant to the following detergents: 5.0% TWEEN® 20, 5.0%TRITON® X-100, 0.1% NP-40®, 0.1% CHAPS, and0.2% digitonin. This gel can also be used with1.0 M NaCl or 1.0 M urea.•Do not use the gel in the presence of SDS, deoxycholate, or guanidine HCl. This is not acomprehensive list of interfering substances. Forgeneral guidance, a Reagent Compatibility Tableis provided on page 5.A.Isolation of FLAG® Fusion Proteins fromYeast BJ3505 Broth(Proceed to Section B, Column Set-Up, if you are not working in yeast.)1.Grow 50 mL of culture in YP expression medium(1% dextrose, 3% glycerol, 1% yeast extract, and 2% peptone) using optimized conditions forsecreted expression of FLAG® fusion protein.2.Divide the culture into two sterile plasticcentrifuge tubes and centrifuge at 10,000 × g for5 minutes.3.Pipette 20 mL of supernatant from eachcentrifuged tube into fresh sterile plasticcentrifuge tubes. Carefully pipette thesupernatant without transferring the cell pellet. 4.Centrifuge the supernatant at 15,000 × g for15 minutes.5.Pipette 15 mL from each centrifuged tube into asterile plastic 50 mL storage tube. Keep on ice.Note: If you do not wish to store your protein,you may proceed directly to Step 7.6. Storage of the FLAG ® fusion protein (Optional): • Sterile-filter the centrifuged supernatant by passing it through a 0.45 µm filter. •15 mL per filter may be processed before back pressure is too high from particulates clogging the filter.•Centrifuged culture broth from YP4 media cannot be sterilized using 0.45 µm filters, since they will become clogged.•The filtered supernatant may be stored on ice for up to a week before degradation of the FLAG ® fusion protein begins to occur.7. Buffer exchange into TBS/Ca buffer (50 mM Tris,pH 7.4, with 0.15 M NaCl and 10 mM CaCl 2) to insure high reproducible binding of the FLAG ® fusion protein. Two methods are available:•Add 9 mL of centrifuged culture broth to 1 mL of 10× TBS/Ca (0.5 M Tris, pH 7.4, with 1.5 M NaCl and 100 mM CaCl 2).•Take 10 mL of centrifuged culture broth and buffer exchange into TBS/Ca on a Sephadex ® G-25 desalting column.B. Column Set-Up1. Place the empty chromatography column on afirm support. 2. Attach a drainage tube to the column to controlthe flow rate. Limit the length of tubing to 25 cm. 3. Remove the top and bottom tabs and rinse thecolumn twice with TBS (50 mM Tris with 150 mM NaCl, pH 7.4). Allow the buffer to drain from the column and leave residual TBS in the column to aid in packing the ANTI-FLAG ® M1 Affinity Gel. C. Packing the Column1. Thoroughly suspend the vial of ANTI-FLAG ® M1Affinity Gel to make a uniform suspension of the gel beads. 2. Immediately transfer the suspension to thecolumn. 3. Allow the gel bed to drain and rinse the vial withTBS. 4. Add the rinse to the column and allow the columnto drain again. The gel bed will not crack when excess solution is drained under normalcircumstances, but do not let the gel bed dry. D. Washing the ColumnWash the gel by loading three sequential 5 mL aliquots of 0.1 M glycine HCl, pH 3.5, followed by three sequential 5 mL aliquots of TBS. Avoiddisturbing the gel bed while loading. Let each aliquot drain completely before adding the next. Do not leave the column in glycine HCl buffer for longer than 20 minutes.E. Binding FLAG ® Fusion Proteins to the Column1. Proper binding of FLAG ® fusion proteins to theANTI-FLAG ® M1 affinity column requires 0.15 M sodium chloride at pH 7.0 as well as the presence of calcium. Before loading the lysate or culture supernatant onto the ANTI-FLAG ® M1 affinity column, be sure that it contains at least 1 mM CaCl 2.Note : If the sample contains particulate material, centrifuge or filter prior to applying to the column. Viscous samples should be treated with DNase or sonicated prior to loading on the column.2. Load the supernatant onto the column undergravity flow. Fill the column completely several times or attach a 12 mL column reservoir prior to loading for larger volumes. Depending upon the protein and flow rate, all of the antigen may not bind. Multiple passes over the column will improve the binding efficiency. 3. Wash the column three times with 12 mL aliquotsof TBS/Ca (TBS containing 1 mM CaCl 2). F. Elution of FLAG ® Fusion ProteinsThree protocols are provided here as suggested protocols for elution of FLAG ®-tagged proteins from this ANTI-FLAG ® M1 Affinity Gel.1. Elution of FLAG ® Fusion Proteins by Acid Elutionwith Glycine:•Elute the bound FLAG ® fusion protein from the column with six 1 mL aliquots of 0.1 M glycine HCl, at pH 3.5, into vials containing 15-25 µL of 1 M Tris, pH 8.0.•Do not leave the column in glycine-HCl buffer for longer than 20 minutes.2. Elution of FLAG ® Fusion Proteins by EDTAChelating Agent:•Incubate the column with 1 mL of TBS/EDTA (TBS containing 2 mM EDTA) for 30 minutes to chelate the calcium ions.•Follow with 1 mL aliquots of TBS/EDTA at 10-minute intervals. Six elution aliquots are usually sufficient to elute the FLAG ® fusion protein.3. Elution of FLAG ® Fusion Proteins by Competitionwith FLAG ® Peptide:• Allow the column to drain completely. •Elute the bound FLAG ®-tagged protein by competitive elution with five one-columnvolume aliquots of a solution with 100 µg/mL FLAG ® peptide (Cat. No. F3290) in TBS.G. Storing the Column1. Wash the column three times with 5 mL of TBS/A(TBS containing 0.02 % sodium azide). 2. Then add another 5 mL of TBS/A. 3. Store at 2-8 °C without draining. H. Recycling the Column1. It is recommended the column be regeneratedimmediately after use by washing with three 5 mL aliquots of glycine HCl, pH 3.5. 2. The column should be immediatelyre-equilibrated in TBS until the effluent is at neutral pH. General Notes1. When E. coli periplasmic extracts are applied tothe column, it may be possible to reuse the column as many as 20 times. 2. When E. coli crude cell extracts are applied to thecolumn, the column may be reused 3 times before loss of binding capacity is observed. 3. The number of cycles observed will be dependenton variables such as sample condition. 4. Do not leave the column in glycine-HClbuffer for longer than 20 minutes .References1. Terpe, K., Appl. Microbiol. Biotechnol., 60(5),523-533 (2003). 2. Einhauer, A., and Jungbauer, A., J. Biochem.Biophys. Methods , 49(1-3), 455-465 (2001). 3. Chubet, R.G., and Brizzard, B.L., BioTechniques ,20(1), 136-141 (1996). 4. Munjal, Neera, “Bioprocessing of microalgae C.reinhardtii for production and purification of single chain antibody fragment”. Texas A&M University, M.S. thesis, p. 24 (December 2014). 5. Quinones, Kathryn Marie, “Bioprocessing ofrecombinant proteins produced in the chloroplast of Chlamydomonas reinhardtii ”. Texas A&M University, M.S. thesis, p. 32 (August 2015). 6. DeRango-Adem, Eva Francesca, “MacromolecularInteractome of Tetrahymena CHD FamilyChromatin Remodelers ”. University of Toronto, M.Sc. thesis, p. 50 (October 2017).7. Heymann, Gregory Seth, “C-Terminus of HSP70Interacting Protein as a Regulator of Parkin Translocation”. University of Toronto, M.S c. thesis, p. 22 (2019). 8. Jabbar, Javard, “POLR2A/RPB1 subunit of RNApolymerase II interacts with NTD-MED14containing core mediator complex to facilitate basal and activator driven transcription”. Bilke nt University, M.Sc. thesis, p. 21 (June 2020). 9. Direkze, Shamindra Gerald, “Characterisation ofTranscriptional Mediator Subunit, MED17 and its Regulation by Cyclins”. University of London, Ph.D. dissertation, p. 120 (2006). 10. Cabrera, Ilva Esther, “The Role of G ProteinSignaling Components in Growth and Development of the Filamentous Fungus, Neurospora crassa ”. University of CaliforniaRiverside, Ph.D. dissertation, p. 149 (December 2015).11. Ning, Wenjing, “Functionalized membranes forprotein purification and proteolysis prior to mass spectrometry analysis”. Michigan State University, Ph.D. dissertation, p. 28 (2016). 12. Ragazzini, Roberta, “Identification of a tissue-specific cofactor of polycomb repressive complex 2”. Université Pierre et Marie Curie, Ph.D. dissertation, p. 106 (2017). 13. DiChiara, Andrew Stephen, “Type I CollagenProteostasis”. Massachusetts Institute ofTechnology, Ph.D. dissertation, p. 149 (February 2018). 14. Kulkarni, Sayali Vishwas, “Bioprocessing ofmicroalgae for extraction of high-value pro ducts”. Texas A&M University, Ph.D. dissertation, p. 101 (August 2018). 15. Ravi, Ayswarya, “Evaluation of mixed -modechromatography resins for isolation ofrecombinant therapeutic proteins”. Texas A&M University, Ph.D. dissertation, p. 43 (August 2019). 16. Laurien, Lucie Henriette, “The role of RIPK1auto-phosphorylation at S166 in cell death and inflammatory signaling ”. Universität zu Köln, Ph.D. dissertation, p. 29 (2021).Troubleshooting GuideProblem Possible Cause SolutionNo signal is observed. FLAG® fusion proteinis not present inthe sample.•Make sure the protein of interest contains the FLAG®-tag by immunoblotor dot blot analyses.•Prepare fresh lysates. Avoid using frozen lysates.•Use appropriate protease inhibitors in the lysate or increase theirconcentrations to prevent degradation of the FLAG® fusion protein. Washes are too stringent.•Reduce the number of washes.•Avoid adding high concentrations of NaCl to the mixture.•Use solutions that contain less or no detergent.Incubation times areinadequate.Increase the incubation times with the affinity resin (from several hours toovernight).Interfering substance ispresent in sample.•Lysates with high concentrations of dithiothreitol (DTT),2-mercaptoethanol, or other reducing agents may destroy antibodyfunction, and must be avoided.•Excessive detergent concentrations may interfere with the antibody-antigen interaction. Detergent levels in buffers may be reduced bydilution.Detection system isinadequate.If Western blotting detection is used:•Check primary and secondary antibodies using proper controls to confirmbinding and reactivity.•Verify that the transfer was adequate by staining the membrane withPonceau S.•Use fresh detection substrate or try a different detection system.Background is too high. Proteins bind nonspecificallyto the ANTI-FLAG®monoclonal antibody, theresin beads, or themicrocentrifuge tubes.•Pre-clear lysate with Mouse IgG-Agarose (Cat. No. A0919) to removenonspecific binding proteins.•After suspending beads for the final wash, transfer entire sample to aclean microcentrifuge tube before centrifugation.Reagent Compatibility TableReagent Effect CommentsChaotropic agents (for example, urea, guanidine HCl) Denatures the immobilizedM1 antibody•Do not use any reagent that contains chaotropic agents, since chaotropicagents will denature the M1 antibody on the resin and destroy its abilityto bind the FLAG® fusion proteins.•If necessary, low concentrations of urea (1 M or less) can be used.Reducing agents (such as DTT, DTE, 2-mercapto-ethanol) Reduces the disulfide bridgesholding the M1 antibodychains togetherDo not use any reagent that contains reducing agents, since reducing agentswill reduce the disulfide linkages in the M1 antibody on the resin and destroyits ability to bind FLAG® fusion proteins.TWEEN® 20, 5% or less Reduces nonspecific proteinbinding to the resin May be used up to recommended concentration of 5%, but do not exceed.TRITON™ X-100, 5% or less Reduces nonspecific proteinbinding to the resin May be used up to recommended concentration of 5%, but do not exceed.IGEPAL® CA-630, 0.1% or less Reduces nonspecific proteinbinding to the resin May be used up to recommended concentration of 0.1%, but do not exceed.CHAPS, 0.1% or less Reduces nonspecific proteinbinding to the resinMay be used up to recommended concentration of 0.1%, but do not exceed.Digitonin, 0.2% or less Reduces nonspecific proteinbinding to the resin May be used up to recommended concentration of 0.2%, but do not exceed.Sodium chloride, 1.0 M or less Reduces nonspecific proteinbinding to the resin byreducing ionic interactionsMay be used up to recommended concentration of 1.0 M, but do not exceed.Sodium dodecyl sulfate Denatures the immobilizedM1 antibody•Do not use any reagent that contains sodium dodecyl sulfate in theloading and washing buffers, since sodium dodecyl sulfate will denaturethe M1 antibody on the resin and destroy its ability to bind FLAG® fusionproteins.•Sodium dodecyl sulfate is included in the sample buffer for removal ofprotein for immunoprecipitation. However, after contact with sodiumdodecyl sulfate, the resin cannot be reused.0.1 M glycine HCl, pH 3.5Elutes FLAG® protein fromthe resinDo not leave the column in glycine HCl for longer than 20 minutes. Longerincubation times will begin to denature the M1 antibody.Deoxycholate Interferes with M1 binding toFLAG® proteins Do not use any reagent that contains deoxycholate, since deoxycholate will inhibit the M1 antibody from binding to FLAG® fusion proteins.The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.NoticeWe provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve ourcustomers of their own responsibility for checking the suitability of our products for the envisaged purpose. The information in this document is subject to change without notice and should not be construed as acommitment by the manufacturing or selling entity, or an affiliate. We assume no responsibility for any errors that may appear in this document.Technical AssistanceVisit the tech service page at /techservice .Standard WarrantyThe applicable warranty for the products listed in this publication may be found at /terms .Contact InformationFor the location of the office nearest you, go to /offices .。

文章编号:1007-8738(2002)04-329-03N otch 信号途径效应分子H ES 1的原核表达及其多克隆抗体的制备与鉴定刘　飞,金伯泉,朱　勇,欧阳为明,张新海,谢　鑫,陈丽华(第四军医大学免疫学教研室,陕西西安710032)收稿日期:2002-01-11;　修回日期:2002-03-15基金项目:国家自然科学基金资助,N o.C39800159,C39880011作者简介:刘　飞(1972-),男,陕西咸阳市人,博士.西安市长乐西路17号,T el.(029)3374531Email :immuneol @关键词:N otch 途径;HES1分子;抗体;原核表达中图分类号:R735.1 文献标识码:A摘　要:目的　原核表达HES1分子,并制备其特异性多克隆抗体,以研究该分子在肿瘤细胞中的表达情况。

方法　诱导表达G ST 2HES1融合蛋白和G ST ,经谷胱甘肽2Sepharose 4B 亲和纯化后,用G ST 偶联预激活的Sepharose 4B ,以G ST 2HES1为免疫原制备兔抗血清。

得到的抗血清经G ST 2Sepharose 4B 吸收抗G ST 成分,以间接E LIS A 和Western blot鉴定抗体特异性,以Western blot 分析胃癌、结肠癌及其相应非癌变组织中HES1的表达水平。

结果　成功地表达并纯化了G ST 2HES1融合蛋白。

制备的抗HES1多克隆抗体具有很高的特异性,与G ST 或大肠杆菌成分无交叉反应,用于进行天然HES1分子的Western blot 检测时效果良好。

初步检测发现,胃癌和结肠癌中HES1的表达水平明显高于相应的正常组织。

结论　通过原核表达并纯化HES1,成功地制备了该分子的特异性多克隆抗体,初步应用效果满意。

Prokaryotic expression of HES 1and prepara 2tion of its polyclonal antibodyLIU Fei ,JIN Bo 2quan ,ZHU Yong ,OUY ANG Wei 2ming ,ZH ANG Xin 2hai ,XIE Xin ,CHEN Li 2huaDepartment of Immun ology ,Basic Medicine C ollege ,F ourth M ilitary Medical University ,X i ’an 710032,Shaanxi Province ,China K eyw ord :notch signal pathway ;HES1;antibody ;prokary otic ex 2pressionAbstract :Aim T o express HES1and prepare its specific antibod 2ies for investigation of its expression in tum ors.Methods G ST 2HES1fusion protein and G ST were induced to express in E.Coli ,then were purified with glutathione 2Sepharose 4B.Purified G ST 2HES1was used to immunize rabbit and purified G ST was coupled with C NBr 2activated Sepharose 4B.The rabbit anti 2HES1serum was harvested and the anti 2G ST antibodies in the serum were abs orbed with G ST 2Sepharose 4B.The specificity of the ployclonal anti 2HES1antibody was determined by indirect E LIS A and Western blot.The expression of HES1in gastric cancer ,colon cancer and correspond 2ing non 2tum or tissues were detected by Western blot.R esults G ST 2HES1fusion protein was expressed and purified success fully.Prepared ployclonal anti 2HES1antibody could recognize native HES1and showed high specificity against HES1without cross 2reaction with G ST or E.Coli component in E LIS A and Western blot.Preliminary western blot result indicated that the expression of HES1in gastric cancer and colon cancer was significantly higher than that in corre 2sponding non 2tum or tissues.Conclusion HES1success fully ex 2pressed in E.coli and purified.Bloyclonal anti 2HES1antibody with high specificity is prepared and applied success fully in preliminary investigation. DNA 结合蛋白(hairy enhancer of split ,HES )是一类含有特征性α螺旋2环2螺旋(αH LH )结构的DNA 结合蛋白[1]。

·论著·2012年11月第9卷第32期中国医药导报CHINA MEDICAL HERALD[基金项目]广东省广州市科技支撑计划项目（项目编号：2009Z1-E011）。

[通讯作者]朱慧兰（1966.9-），女，中共党员，主任医师，硕士生导师，广州市卫生局局管优秀科技人才，现任广州市皮肤病防治所副所长，主要从事皮肤病方面的研究。

CD20抗原是一种B 细胞分化抗原，仅位于前B 细胞和成熟B 细胞，而在造血干细胞、血浆细胞和其他正常组织中不表达[1]，是B 细胞靶向治疗的理想作用位点。

抗CD20单克隆抗体（mAb ）以B 细胞表面分子CD20为靶向，其用于治疗B 细胞淋巴瘤已经获得了令人鼓舞的疗效[2]。

近年来也越来越多地应用到自身免疫性疾病的治疗，包括慢性荨麻疹（chronic urticaria ，CU ）的治疗[3]。

慢性荨麻疹是一种常见皮肤病，目前治疗效果不佳，且其发病机制较为复杂，至今尚未完全清楚[4-8]。

虽有研究表明将B 细胞靶向治疗应用于慢性荨麻疹具有良好疗效[9-11]，但目前资料有限，对于具体的作用机制尚不清楚。

为了寻找更为有效安全的用于治疗慢性荨麻疹的CD20抗体药物，本研究在前期筛选获取抗CD20单克隆细胞株的基础上，拟克隆其重链和轻链的基因片段，构建重组真核表达载体，并最终表达、纯化具有细胞结合活性的抗CD20嵌合抗体，为下一步治疗慢性荨麻疹的研究打下基础。

1材料与方法1.1实验材料杂交瘤细胞株、TOP10（大肠埃希菌）、293E 细胞、NS-1细胞为本实验室保存。

RNA 提取试剂盒、RT-PCR 试剂盒购自美国Promega 公司；质粒提取试剂盒和胶回收试剂盒购于康为世纪公司；DNA 连接酶、限制性内切酶、Ex Taq 酶、DNA Marker 购于大连宝生物公司；转染试剂Lipofectamine 2000、RPMI 1640培养基、DMEM 培养基购自Invitrogen 公司；改良型快速杂交瘤ＣＤ２０嵌合抗体的构建、表达及纯化的实验研究梁碧华李润祥马少吟龚业青李薇毕超朱慧兰广东省广州市皮肤病防治所，广东广州510095[摘要]目的构建CD20嵌合抗体，检测其活性并纯化该嵌合抗体。