Anti-DYKDDDDK Affinity Beads
BeaverBeadsTM Streptavidin 链霉亲和素磁珠说明书
链霉亲和素磁珠（BeaverBeads TM Streptavidin ）产品简介链霉亲和素-生物素（SA-Biotin ）系统具有极高的结合亲和力（Kd=10^-15），在生物领域具有广泛的应用。
BeaverBeads TM Streptavidin 采用海狸专利的蛋白偶联技术将SA 共价连接于固相载体表面，可高效结合生物素化抗体、核酸、蛋白等配体分子。
本产品采用超顺磁性微球，粒径均一、形貌规整，有利于方便、快捷地捕获目标分子以及实现磁性分离。
本产品可配套自动化设备进行高通量操作。
产品信息产品信息 SA 磁珠（300 nm ）生物素化IgG 结合性能 20μg/mg 磁珠 磁珠浓度 10 mg/mL 磁珠表面 亲水基团保存溶液 1×PBS ，含0.1%（w/v ）BSA ，0.1%（v/v ）proclin-300 保存条件 2-8 ℃ 保质期2年产品应用范围适用于体外诊断体系磁微粒化学发光免疫诊断操作流程（本操作以两步双抗体夹心CLIA 法为例）1. 使用前准备1.1. 检测试剂：包括捕获抗体、待测物标准品、待测样品、酶标记抗体、底物液等，使用前平衡至室温1.2. Washing buffer ：用户根据需求配制洗液，使用时平衡至室温 1.3. 化学发光96孔平底微孔板1.4. 96孔平底微孔板磁性分离器：可选用海狸磁性分离器，Cat.No.60302 1.5. 漩涡振荡器 1.6. 真空吸液泵 1.7. 化学发光仪 1.8. 移液器2. 磁微粒化学发光免疫诊断操作流程2.1.调整磁珠至合适浓度（建议0.8mg/ml ），将磁珠置于漩涡振荡器上20 s ，振荡重悬磁珠。
用移液器移取50 μL 磁珠至96孔板中，磁性分离，用移液器吸去上清液，从磁性分离器上取下96孔板。
2.2. 每孔加入100 μL 生物素化捕获抗体，充分震荡重悬磁珠，37℃恒温箱中孵育15min 后，磁性分离，用移液器吸去上清液，从磁性分离器上取下96孔板。
FLAG M浓缩纯化试剂包说明书
Technical BulletinFLAG® M Purification Kit For mammalian expression systems CELLMM2Product DescriptionEpitope-tagged proteins can be affinity-purified using highly specific antibodies that are raised against their epitope. This is useful for the isolation of recombinant proteins or protein complexes. The use of such antibodies facilitates subsequent biochemical and immunological analysis.The FLAG® epitope system relies on the FLAG®octapeptide(N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C; DYKDDDDK), which allows FLAG® fusion proteins to retain their original conformation and function. The hydrophilic character of FLAG® increases the likelihood that it will be located on the surface of the fusion protein where it is accessible to antibodies. The kit allows rapid and efficient affinity purification of active FLAG® fusion proteins. It can be also used for immunoprecipitation. The affinity purification is performed with ANTI-FLAG®-M2 affinity gel, which is a highly specific monoclonal antibody covalently attached to agarose resin. The use of affinity resin allows efficient binding of FLAG® fusion proteins without the need for preliminary steps and calibrations. The affinity-bound FLAG® fusion proteins can be efficiently eluted from the resin by acidic conditions or by competition with 3X FLAG® peptide. The eluted proteins can be analyzed for their activity, size, post-translational modifications, interactions, and other properties.1-5The FLAG® M Purification Kit includes the CelLytic™ M Cell Lysis Reagent. CelLytic™ M enables efficient and rapid cell lysis, and solubilization of proteins for cells in suspension and adherent cells such that adherent cells do not require scraping from culture dishes. Several publications cite use of the CELLMM2 product in their research protocols.6-10Storage/StabilityThe kit is shipped on wet ice and stored at -20 °C. Upon first thawing, the buffers may be stored at2-8 °C. For recommended storage of the 3X FLAG®peptide (Cat. No. F4799) and the Amino-terminal FLAG-BAP™ fusion protein (Cat. No. P7582), see the Preparation Instructions. CelLytic™ M Cell Lysis Reagent (Cat. No. C2978) may appear cloudy after an extended period of storage. Product performance is unaffected, and it may be used, as is, without further filtration or clarification. ReagentsSufficient reagents are provided for 3-5 affinity purifications through a 1 mL affinity purification column.•CelLytic™ M (Cat. No. C2978): 120 mL•10× Wash Buffer [Component Number W0390;0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl]: 60 mL •Elution Buffer [Component Number E6150; 0.1 M Glycine (pH 3.5)]: 60 mL•3X FLAG® Peptide (Cat. No. SAE0194): 4 mg •ANTI-FLAG® M2-Agarose Affinity Gel (Cat. No.A2220): 1 mL•Amino-terminal FLAG-BAP™ Fusion Protein (Component Number I7582): 40 µg •Polypropylene chromatography column (Cat. No.C2103): 5 eachReagents and Equipment Required(But not provided)•Test tubes•Hamilton® syringe (such as Cat. No. 24539) or a Pasteur pipette (such as Cat. No. S6268) •Shaker•Centrifuge•Microcentrifuge, Eppendorf® 5417R or equivalent •Dulbecco’s Phosphate Buffered Saline (PBS), such as Cat. No. D8537•Protease inhibitor cocktail (such as Cat. No.P8340; see Related Products for examples ofother inhibitor cocktail products)•SIGMA FAST™ pNPP substrate tablets set (such as Cat. No. N1891), or pNPP liquid substrate system (such as Cat. No. N7653)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.Preparation Instructions• Defrost solutions: Be sure all kit solutions are defrosted and are mixed to a homogenous form. •5 µg/µL 3X FLAG ® peptide solution: The 3X FLAG ® peptide (N-Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C) is acidic. To dissolve it properly, add 160 µL of 10× Wash Buffer to 4 mg of 3X FLAG ® peptide. When the peptide is completely dissolved, add 640 µL of distilled water to the sample. Mix well and store aliquots at -20 °C. •Dilute FLAG-BAP ™ fusion protein: For control reactions, dilute a portion of the FLAG-BAP ™ fusion protein solution to a concentration of 50 ng/µL with 1× Wash Buffer. The dilutedsolution is stable at –20 °C for about two months.ProceduresGeneral notes•See the Certificate of Analysis for A2220 for the lot-specific binding capacity of the ANTI-FLAG ® M2 affinity resin.•The ANTI-FLAG ® M2 affinity resin is stored in 50% glycerol with buffer. The glycerol must beremoved just prior to use, and the resin should be equilibrated with 1× Wash Buffer. Theequilibration can be performed either at room temperature or at 2-8 °C.•To check reagent compatibility with theANTI-FLAG ® M2 affinity gel, see the Reagent Compatibility Table.•For an immunoprecipitation procedure, see the Technical Bulletin for the FLAG ®Immunoprecipitation Kit (Cat. No. FLAGIPT1) on the Web site. Use the CelLytic ™ M reagent component of this kit as a lysis buffer.Cell LysisNotes for cell lysis •The volume of CelLytic ™ M reagent to add to the cells varies with the cell size and proteinconcentration required. In general, 125 µL of CelLytic ™ M are recommended for 106-107 cells. For adherent cells, the plate size will dictate the amount of buffer covering the plate surface.Suggested working volumes are: 500-1000 µL for a 10 cm plate. 200-400 µL for a 3.5 cm plate.•In general, to avoid protein degradation, it isrecommended that Protease Inhibitor Cocktail be added to the CelLytic ™ M reagent.•Lysate preservation requires low temperatures. Therefore, store the lysate at -70 °C for long-term storage. 1. Wash cells and treat with cell lysis buffer.a. For adherent cells: • Remove the growth medium from the cells to be assayed.• Rinse the cells once with PBS buffer, being careful not to dislodge any of the cells. • Discard PBS.• Add CelLytic ™ M reagent.b. For cells in suspension:• Collect the cells into an appropriate conical centrifuge tube.• Centrifuge for 5 minutes at 450 × g. • Decant the supernatant and discard. •Wash the cells once by resuspending the cell pellet with PBS and centrifuge for 5 minutes at 450 × g .• Decant supernatant and discard. •Resuspend the cell pellet in CelLytic ™ M reagent.2. Incubate the cells for 15 minutes on a shaker.3. Collect cell lysate.• For adherent cells: collect cells. •For cells in suspension: skip to Step 4.4. Centrifuge the lysed cells for 10 minutes at12,000-20,000 × g to pellet the cellular debris. 5. Remove the protein-containing supernatant to achilled test tube.• For immediate use, keep on ice. •Otherwise, store the protein as a frozen solution.Affinity Purification (column)General notes on protein purificationFLAG ® fusion protein can be affinity-purified either on a column or by batch format. The column format works well when there is no substantial difference between the volume of material to load onto the column and the amount of resin being used. Forlarger volumes, the batch format is recommended to capture quickly the target protein from a large volume of extract.If you work with a lysate volume up to ~ 6 mL and you do not have equipment for buffer loading under a controlled flow, then for best results, you can use the column closed at its two ends and work according to the batch format.Pre-equilibrate the column and buffers. Perform the purification at room temperature. If there is a problem with proteases, perform columnchromatography at 2-8 °C, or add Protease Inhibitor Cocktail to the elution solution.Cellular debris and particulate matter can clog the column and must be removed prior to purification, especially if the sample has been defrosted.A large amount of insoluble material may requirecentrifugation (10,000-20,000 × g for 15 minutes) for removal. The protein extract should also be filtered with a 0.45 µm filter to remove any remaining cells and particulates.Highly viscous samples containing chromosomal DNA or RNA can also clog the column. Viscous samples should be sonicated or treated with nuclease toreduce viscosity. FLAG-BAP ™ positive control proteins can be used to verify the functionality of the gel. Resin preparation1. Place the chromatography column on a firmsupport. 2. Rinse the empty column with 0.5 mL of 1× WashBuffer. Allow the buffer to drain from the column and leave residual Wash Buffer in the column to aid in packing the resin. 3. Thoroughly suspend the resin by gentle inversion.Make sure the ANTI-FLAG ® M2 affinity gel is a uniform suspension of gel beads. Remove the required amount of resin for use. 4. Immediately transfer the suspension to thecolumn. 5. Allow the gel bed to drain and rinse the pipetteused for the resin aliquot with 1× Wash Buffer. The 50% glycerol buffer will flow slowly and the flow rate will increase during the equilibration. 6. Add the rinse to the top of the column and allowto drain again. The gel will not crack when excess solution is drained under normal circumstances, but do not let the gel bed run dry. 7. Load three column volumes of Elution Buffer. Letthe buffer drain completely. Avoid disrupting the gel bed while loading. Do not leave the column in loading buffer longer than 20 minutes. This step is a mock elution for removal of residual impurities off the column. Wash the resin with five column volumes of 1× Wash Buffer, or until the eluent is at a neutral pH to equilibrate the resin for use. Do not let the bed run dry. Allow a small amount of buffer to remain on top of the column. Do not allow the resin to remain in 1× Wash Buffer for extended periods of time (>24 hours) unless an antimicrobial agent (such as sodium azide) is added to the buffer.Column chromatography1. Load the sample onto the column under gravityflow. Fill the column completely several times, or attach a column reservoir prior to loading for larger volumes. In cases when the FLAG ® fusion protein is not completely bound (depending on the specific protein and on the loading flow rate), multiple passes over the column will improve the binding efficiency. 2. Usually the sample loading step requires a slowflow to allow binding of the fusion protein to the affinity resin. If the sample volume is up to ~ 6 mL, it can be loaded in a batch mode byincubation of the resin and sample solution in the column, under a gentle rotation. 3. Wash the column with 10-20 column volumes of1× Wash Buffer. This should remove any proteins that are not bound to the M2 antibody. Allow the column to drain completely. Elution of FLAG ® Fusion ProteinsSelect one of the two following elution procedures: 1. Acid Elution with Glycine:•Elute the bound FLAG ® fusion protein from the column with six 1 mL aliquots of Elution Buffer into vials which contain 50-100 µL of10× Wash Buffer/1 mL of eluent or 15-25 µL of 1 M Tris (pH 8).• Do not leave the column in Elution Buffer for longer than 20 minutes.•Re-equilibrate column to neutral pH as soon as possible after elution.2. Competition with 3X FLAG ® Peptide: Elute thebound FLAG ® fusion protein by competitiveelution with five one-column volumes of a solution containing 150-200 ng/µL of 3X FLAG ® peptide (Cat. No. F4799) in 1× Wash Buffer. Notes •The elution can be performed stepwise by a sequential addition and collection of a defined volume of Elution Buffer.•When the elution step requires an extendedincubation of the resin with the Elution Buffer or 3X FLAG ® peptide, incubate under a gentle rotation.• Do not leave the column in Elution Buffer for longer than 20 minutes.•When elution efficiency is low, the concentration of 3X FLAG ® in suspension can be increased, or alternatively, a salt can be added to the Elution Buffer.Batch Absorption of FLAG ®Fusion Proteins using ANTI-FLAG ® M2 Affinity GelThis method provides a quick and efficient way to purity FLAG ® fusion proteins from a dilute solution. It eliminates the time-consuming columnchromatography step of placing a large volume of solution through a small amount of resin.1. Re-suspend the equilibrated resin (see Resinpreparation) in 1× Wash Buffer and add to the protein extract. 2. Incubate the protein extract with the ANTI-FLAG ®M2 affinity gel for ~1 hour with gentle mixing to capture the FLAG ® fusion proteins. Mixing should be done on either an overhead mixing device or a platform shaker. Do not use a magneticstirring system, as this will destroy the resin beads. This step can be performed at 2-8 °C or at room temperature. The incubation time can range from 30 minutes to several hours. If the incubation is longer than 3 hours, proteaseinhibitors and antimicrobial substances should be added to prevent microbial growth and/or proteolysis. 3. Once the binding step is complete, collect theresin from the container. The resin can be collected by centrifugation (1,000 × g for 5 minutes) or by filtration. 4. Wash the resin with 1× Wash Buffer to remove allof the non-specific proteins. This may be done in the column format by passing fresh buffer through the column until no further proteinelutes. The eluted protein from the resin can be monitored by measuring the absorbance of the eluent at 280 nm. Continue washing the resinuntil the absorbance of the wash solution from the column is < 0.05 vs. a wash solution blank. 5. The FLAG ® proteins can be eluted from the resineither by low pH (Elution Buffer) or bycompetition with the 3X FLAG ® peptide. Follow the elution steps under Elution section. 6. The resin can be recycled and stored as per theRecycling the Column and the Storing the Column sections.Recycling the ColumnIt is recommended that the column be regenerated immediately after use by washing with three column volumes of elution buffer. The column should be immediately re-equilibrated in 1× Wash Buffer until the eluent is at neutral pH.Storing the ColumnWash the column with ten column volumes of1× Wash Buffer containing 50% glycerol and 0.02% sodium azide. Then add another 5 mL of buffered glycerol containing 0.02% sodium azide. Store at 2-8 °C or -20 °C without draining.The number of cycles obtained will be dependent on variables such as sample condition and proteases.Alkaline Phosphatase analysisThe FLAG-BAP ™ protein serves as a functional control for immunoaffinity chromatography andimmunoprecipitation with ANTI-FLAG ® M2 affinity gel. When using the control protein, we recommend a small-scale detection.Run the samples and controls on an SDS-PAGE gel. The FLAG-BAP ™ fusion protein has a molecular mass of 49.3 kDa. It migrates as a 45-55 kDa band by SDS-PAGE depending on the electrophoresis conditions.Immunoblot the gel with ANTI-FLAG ® orAnti-Bacterial Alkaline Phosphatase (BAP) antibodies. Alternatively, stain the gel with a staining method such as silver staining or Coomassie ® blue.An alternative option is to detect the BAP presence by enzymatic activity. SIGMA FAST ™ pNPP substratetablets set (Cat. Nos. N1891 or N2770) or pNPP liquid substrate system (Cat. No. N7653) are recommended for the detection of BAP activity.References1. Brizzard, B.L. et al., BioTechniques , 16(4),730-734 (1994). 2. Knappik, A., and Plückthun, A., BioTechniques ,17(4), 754-761 (1994). 3. Chiang, C.M., and Roeder, R.G., Pept. Res ., 6(2),62-64 (1993). 4. Ausubel, F.M. et al. (eds.), Current Protocols inMolecular Biology . John Wiley & Sons, Inc. (New York City, NY), pp. 10.15.1-10.16.29 (1998). 5. Harlow, E., and Lane, D., Antibodies, A LaboratoryManual . Cold Spring Harbor Laboratory Press, (Cold Spring Harbor, NY), pp. 514-517, 541-542, 547-549 (1988).Troubleshooting GuideReagentEffectCommentsChaotropic agents (such as urea, guanidine HCl) Denatures the immobilized M2 antibodyDo not use any reagent that contains these types of components, sincechaotropic agents will denature the M2 antibody on the resin and destroy its ability to bind the FLAG ® fusion proteins. Low concentrations of urea (1 M or less) can be used.Reducing agents (such as DTT, DTE, 2-mercaptoethanol) Reduces the disulfide bridges holding the M2 antibody chains together Do not use any reagent that contains these types of components, sincereducing agents will denature the disulfide linkages in the M2 antibody on the resin and destroy its ability to bind the FLAG ® fusion proteins.TWEEN ® 20, 5% or lessReduces non-specific protein binding to the resinMay be used up to recommended concentration of 5%, but do not exceed.TRITON ® X-100, 5% or lessReduces non-specific protein binding to the resinMay be used up to recommended concentration of 5%, but do not exceed.Igepal ® CA-630, 0.1% or lessReduces non-specific protein binding to the resinMay be used up to recommended concentration of 0.1%, but do not exceed.CHAPS, 0.1% or lessReduces non-specific protein binding to the resinMay be used up to recommended concentration of 0.1%, but do not exceed.Digitonin, 0.2% or lessReduces non-specific protein binding to the resinMay be used up to recommended concentration of 0.2%, but do not exceed.Sodium chloride, 1.0 M or lessReduces non-specific protein binding to the resin by reducing ionic interactionsMay be used up to recommended concentration of 1.0 M, but do not exceed.Sodium dodecyl sulfate (SDS)Denatures the immobilized M2 antibodyDo not use any reagent that contains this detergent in the loading and washing buffers, since SDS will denature the M2 antibody on the resin and destroy its ability to bind the FLAG ® fusion proteins. SDS is included in the sample buffer for removal of protein for immunoprecipitation, but the resin cannot be reused.0.1 M glycine HCl, pH 3.5 Elutes FLAG ® protein from the resinDo not leave the column in glycine HCl for longer than 20 minutes. Longer incubation times will begin to denature the M2 antibody.DeoxycholateInterferes with M2 binding to FLAG ® proteins Do not use any reagent that contains this detergent, since deoxycholate will inhibit the M2 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.References (continued)6. Jin, G. et al., EMBO J., 30(11), 2281-2293(2011). 7. Jeng, M.Y. et al ., J. Exp. Med., 215(1), 51-62(2017). 8. Fu, L. et al., Nucleic Acids Res., 47(7),3568-3579 (2019). 9. Zhang, Z. et al., Cell Rep., 29(1), 49-61.e7(2019). 10. Shi, Y. et al., FASEB J., 34(1), 208-221 (2020).Related Products•Mammalian FLAG ® expression vectors, such as Cat. Nos. E8770, OGS1142, OGS1248, OGS1176, OGS3407, OGS1264, OGS3423, OGS1124, OGS1084, OGS625, OGS624, and OGS618 • Mammalian sequencing primers, such as Cat. No. P5350• Monoclonal ANTI-FLAG ® M1, purified immunoglobulin: Cat. No. F3040• ANTI-FLAG ® M1 Agarose Affinity Gel: Cat. No. A4596• Monoclonal ANTI-FLAG ® M2, purified immunoglobulin: Cat. No. F3165• Monoclonal ANTI-FLAG ® M2, affinity isolated antibody: Cat. No. F1804• Monoclonal ANTI-FLAG ® M5, purified immunoglobulin: Cat. No.F4042 • FLAG ® peptide: Cat. No. F3290• FLAG-BAP control fusion proteins: Cat. Nos. P7582, P7457, and P5975• Glycerol, such as Cat. No. G5516 (for molecular biology)• Deoxyribonuclease I (DNase I), such as Cat. No. D4527•Sodium azide, such as Cat. No. 8032 (BioXtra)• FLAG ® Immunoprecipitation Kit: Cat. No. FLAGIPT1• ANTI-FLAG ® M2-peroxidase conjugate: Cat. No. A8592• ANTI-FLAG ® M2-alkaline phosphatase conjugate: Cat. No. A9469•Protease and phosphatase inhibitor cocktails, such as Cat. Nos. P8215, P2714, P8465, P8340, P9599, P2850, P5726, P0044, MSSAFE, P0001, PIC0002, PIC0004, PIC0005, PIC0006, PPC2020• p -Nitrophenyl Phosphate (pNPP) liquid substrate system: Cat. No. N7653•SIGMA FAST p -Nitrophenyl Phosphate tablets sets: Cat. Nos. N1891 and N2770NoticeWe 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 .。
FLAG标签融合蛋白纯化试剂盒使用说明书
FLAG标签（DYKDDDDK）融合蛋白纯化试剂盒Cat#：KAP0064（本品仅用于科学研究，不可用于诊断及治疗）1.背景信息Flag-Tag（DYKDDDDK），常用于真核蛋白质重组表达标记。
FLAG标签（DYKDDDDK）融合蛋白纯化试剂盒，主要成分是Anti-Flag亲和纯化凝胶（Anti-DYKDDDDK (Flag) Affinity Gel），由高品质的Flag兔多克隆抗体与Sepharose 4B琼脂糖颗粒共价偶联制成，具有高载量（至少为1.2mg protein/ml凝胶），高特异性，性质稳定，可反复使用的特点，可用于Flag 标签融合蛋白的亲和纯化。
2.性能指标应用范围：可用于Met修饰的N端Flag融合蛋白（Met-Flag–Protein），N端Flag融合蛋白（Flag–Protein）和C端Flag融合蛋白（Protein-Flag）的亲和纯化。
载量：1ml Sepharose 4B琼脂糖颗粒，共价偶联800μg Anti-Flag 兔多克隆抗体，可纯化至少1.2mg Flag融合蛋白。
强度：重力柱纯化，可反复使用5次以上。
保存方法：在加入了50%甘油和0.2‰叠氮钠的PBS保存液后，预装纯化柱可于-20℃可保存1年。
3.试剂盒组成50 mM Tris HCl, pH 7.4, 150 mMNaCl, 1 mM EDTA, and 1%Triton X-100（说明见5.3）4.使用方法4.1细胞裂解液制备4.1.1悬浮细胞和半贴壁细胞从细胞培养瓶上吹下来后放入离心管中，1000rpm离心5分钟。
贴壁细胞用细胞刮子轻轻从瓶壁上刮下来，放入离心管中1000rpm离心5分钟。
4.1.2预冷的PBS工作液重悬细胞，1000rpm离心3min，弃上清。
重复一次。
4.1.3根据细胞的量加入相应体积的细胞裂解液，反复吹打后冰上放置10-20min，让细胞充分裂解。
4.1.4用超声破碎仪将细胞裂解液超声，直至细胞裂解液透明，不再粘稠。
Anti-DYKDDDDK Magarose beads
Anti-DYKDDDDK Magarose Beads目录1.产品介绍 (1)2.试剂准备 (1)3.样品纯化 (2)4.试剂兼容性 (3)5.问题及解决方案 (3)6.订购信息及相关产品 (4)1.产品介绍Flag标签是一个由八个亲水氨基酸组成的多肽片段，定位在融合蛋白表面，因此更易与抗体结合以及被肠激酶分解。
Magarose Beads系列产品具有超顺磁性、快速磁响应性、丰富羟基官能团和相对集中的粒径等特点，是医学与分子生物学研究中重要的载体工具。
Anti-DYKDDDDK Magarose Beads是以抗flag(DYKDDDDK)抗体为亲和配体，一步纯化原核、酵母或哺乳动物细胞表达的flag标签融合蛋白。
表1.Anti-DYKDDDDK Magarose Beads产品性能性能指标基质磁性琼脂糖微球配体Anti-DYKDDDDK Antibody结合能力(/ml磁珠)>1mg DYKDDDDK标签蛋白粒径(μm)30-100储存缓冲液PBS，0.01%Tween-20,0.02%NaN3磁珠体积磁珠体积占悬浮液体积的20%储存温度2°C-8°C2.试剂准备2.1样品准备上柱之前要确保样品溶液有合适的离子强度和pH值，可以用平衡液对样品或细胞培养液稀释，或者用平衡液透析。
样品在上样前建议离心或用0.22μm或0.45μm滤膜过滤，减少杂质，提高蛋白纯化效率和防止堵塞柱子。
2.2缓冲液的准备所用水和缓冲液在使用之前建议用0.22μm或0.45μm滤膜过滤。
平衡/洗杂液:50mM Tris,0.15M NaCl,pH7.4洗脱液:0.1M glycine HCl,pH3.0竞争性洗脱液：50mM Tris,0.15M NaCl,100-500ug flag多肽/ml，pH7.4中和液：1M Tris-HCl，pH8.03.样品纯化3.1磁珠预处理将Anti-DYKDDDDK Magarose Beads颠倒数次，保证磁珠完全混匀，取计算量的磁珠悬浮液，转移至离心管中，放置在磁分离器上，静置大约1min，待溶液变澄清后，用移液器吸弃清液。
flag磁珠说明书
flag磁珠说明书Anti-Flag磁珠，也称Anti-Flag免疫磁珠或Flag抗体磁珠，是由高品质的Flag 小鼠单克隆抗体与纳米级氨基磁珠共价偶联而成，可特异性地与动植物或微生物裂解液、血清、腹水等中含有Flag标签的蛋白结合，从而用于带有Flag标签的融合蛋白或其蛋白复合物的免疫沉淀(Immunoprecipitation, IP)、免疫共沉淀(Co-IP)或纯化。
Flag标签(Flag-tag)、Myc标签(Myc-tag)、HA标签(HA-tag)、His标签(His-tag)和GST标签(GST-tag)等是表达载体上最常见的一些标签，通过与这些标签的融合表达可以非常方便地检测目的蛋白及与目的蛋白相互结合的蛋白，也可以非常方便地用于目的蛋白的纯化。
Flag-tag是8个氨基酸残基(DYKDDDDK)组成的多肽，常用的形式有Flag和3X Flag，通过基因重组技术把Flag-tag的核酸序列与目的基因的5’端或3’端连接，就可以最终表达形成Flag-tag的目的蛋白。
Flag-tag具有以下优点：Flag-tag通常不会与目的蛋白相互作用，并且大多数情况下不会影响目的蛋白的功能；Flag-tag作为标签蛋白，后续通过Flag抗体(AF519)、Anti-Flag磁珠或Anti-Flag亲和凝胶(P2271)即可对目的基因的表达、定位及功能进行检测或对目的蛋白进行纯化、免疫沉淀或免疫共沉淀等；融合在N端的Flag标签，可被肠激酶(Enterokinase, EK)切除(DDDK)，从而得到完美的没有标签的目的蛋白。
基于以上优点，Flag标签已被广泛应用于蛋白表达、纯化、鉴定、相互作用和功能等多方面的研究。
Anti-Flag Magnetic Beads (Anti-Flag磁珠)，也被称为Anti-DYKDDDDK Magnetic Beads，可以特异性地结合Flag标签融合蛋白，并可以借助磁力架等磁分离设备非常便捷地应用于带有Flag标签的融合蛋白或其蛋白复合物的免疫沉淀或纯化等实验。
重组表达质粒的构建——原核表达载体选择
重组表达质粒的构建——原核表达载体选择质粒载体是重组蛋白表达的关键工具，其结构如下图。
重组蛋白表达，我们首先要将基因插入到表达载体上，插入的位置为多克隆位点。
质粒载体上有很多的功能元件，这些元件对于蛋白的表达都是至关重要的。
尽管我们经过系统的分析和预测，但是仍有很多蛋白不能顺利表达、表达量很低或者表达状态不好。
这个时候我们需要尝试构建不同的表达载体以期得到最好的效果，这些载体的主要区别是启动子和融合标签的差异。
蛋白表达优化主要工作也就是尝试构建不同融合表达标签，使用不同的宿主表达菌，测试不同的表达条件，筛选出最优表达体系。
常用的融合标签有GST、MBP、Trx、6His、SUMO等，这些标签主要功能是促表达、促可溶、信号标记或助纯化。
福因德生物可以提供以下系列载体以供科研表达研究。
1）促表达/促溶标签2）信标标签3）纯化标签我们选择表达载体的时候不但要考虑蛋白怎么表达成功，更要考虑蛋白怎么纯化出来，纯化的问题主要是考虑纯化标签和酶切位点的选择，下表我们列举了常见的纯化标签和酶切位点。
4）酶切位点以上为原核表达常用的标签和酶切位点,其性质也都作了简要的介绍，各专业网站或专业书籍已对此做详尽解释，科研工作者可根据具体实验设计方案，组合设计以上标签和酶切位点的使用。
特别值得注意的是，选用和设计蛋白酶切位点的时候首要考虑的是序列内部有没有蛋白酶位点，同时要考虑酶切的效率和蛋白酶试剂成本。
一般商业化载体，在标签蛋白与载体多克隆位点之间都设计有酶切位点。
标签可设计在N-端也可在C-端,设计在N-端的优势是,可通过标签高效翻译起始位点带动插入蛋白的表达,可溶性标签的高效表达更可促进蛋白的可溶性表达;同时,大部分的蛋白内切酶的切割位点在C-端,所以标签设计在N-端可将标签切割完全。
在设计标签序列与酶切位点的时候还要考虑N-端稳定性原则，也就是所谓宿主细胞的N-端规则（N-end rule）,这个要避免；同时，还应该检查是否引入了可与别的蛋白相互作用的序列或者蛋白酶切位点。
常见蛋白质标签总结
/bbs/home.php?mod=space&uid =34800&do=blog&id=38530常见蛋白质标签总结（Flag、HA、cMyc、CBP等）Protein tags are peptide sequences genetically grafted onto a recombinant protein. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing. Tags are attached to proteins for various purposes.一、氨基酸标签（含小肽标签）A stretch of amino acids is added to the protein and enables the recovery of the labelled protein by its unique affinity. Usually its easiest to add the tag to either end of the protein to ensure its accessibility and not to disturb the protein folding.1.组氨酸标签（His tag）一般为6个组氨酸，用Ni2+（Cu2+）亲和层析纯化2.FLAG tag ：N-DYKDDDDK-C ，recovered with specific antibody3.HA tag： an epitope derived from the Influenza protein haemagglutinin (HA，禽流感病毒血凝素)，e.g. N-YPYDVPDYA-C，recovery with an HAantibody4.MYC tag： an epitope derived from the human proto-oncoprotein MYC，e.g.N-ILKKATAYIL-C, N-EQKLISEEDL-C，recovery with an MYCantibody5.SBP tag：Streptavidin Binding Peptide，链霉亲合素结合肽，38 amino acidtag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)，更多参考在Sigma6.CBP tag：钙调蛋白结合肽（CBP; 26aa）钙调蛋白结合肽与钙调素结合是Ca2+依赖的，这种结合不受标签所处的位置影响（N端和C端均可），在中性pH条件下使用2mM EGTA可以很方便的将目标蛋白洗脱下来。
FlowComp
Kit ContentsFlowComp ™ Dynabeads ® contains ~1 × 109 (~10 mg) beads/mL in phosphate buffered saline (PBS), pH 7.4, with 0.1% bovine serum albumin (BSA) and 0.02% sodium azide as a preservative. FlowComp ™ Human CD3 Antibody contains monoclonal CD3 antibody in PBS with 0.5% BSA and 0.02% sodium azide. FlowComp ™ Release Buffer contains modified biotin in 0.1% BSA and 2 mM EDTA.Caution: Sodium azide may react with lead and copper plumbing to form highly explosive metal azides.Product DescriptionFlowComp ™ Human CD3 is intended for positive magnetic isolation of CD3+ T cells from human peripheral bloodmononuclear cells (PBMCs). The isolated cells are highly pure, viable, and bead-free (fig. 1). In the first step, FlowComp ™ Human CD3 Antibody is added and binds to the target cells. In the second step, CD3+ T cells, that have bound the specific antibodies, are captured by the FlowComp ™ Dynabeads ®. In the third and last step, the cells are released from the FlowComp ™ Dynabeads ®.Dynabeads ® FlowComp ™ Human CD3Isolation from PBMCFor research use only. Not for human or animal therapeutic or diagnostic use.Catalog no. 11365D Store at 2˚C to 8˚CRev. Date: February 2012 (Rev. 001)Downstream ApplicationsIsolated cells are bead-free and may be used directly in anydownstream application including flow cytometry. The cells readily proliferate in response toDynabeads ® Human T Activator CD3/CD28 and can be measured by incorporating EdU or in a CFSE assay.Required Materials• Magnet (DynaMag ™ portfolio).See /magnets for recommendations.• Mixer allowing tiltingand rotation of tubes (e.g. HulaMixer ® Sample Mixer).• Isolation Buffer:Ca 2+ and Mg 2+ free PBSsupplemented with 0.1% BSA and 2 mM EDTA.Note: BSA can be replaced by human serum albumin (HSA) or 2% FBS/FCS.• Optional: Flow cytometry antibodies. We recommend using anti-CD3clone UCHT-1 from Caltag Medsystems as primary fluorescent antibody for flow staining of cells after isolation. Optional clones: OKT3, HITa3.• Optional: For viability analysis, SYTOX ® Red is recommended.General Guidelines• Visit /cellisolation and follow ourQuickLinks for recommended sample preparation procedures.• Use a mixer that provides tilting and rotation of the tubes to ensure thatbeads do not settle in the tube.• This product should not be used with the MPC ™-1 magnet(Cat. no. 12001D).• Avoid air bubbles (foaming) during pipetting.• Never use less than the recommended volume of beads.• Carefully follow the recommended pipetting volumes and incubationtimes.• Keep all buffers cold.• To avoid unspecific labeling of cells during flow staining, werecommend using gammaglobulin prior to staining with primary fluorescent antibody.• For better purity, repeat the washing step once or transfer the bead-bound cells to a new tube before adding the FlowComp ™ Release Buffer.• All incubations at room temperature can also be performed at2°C to 8°C.ProtocolThis protocol is intended for isolation of bead-free CD14+ monocytes, starting with 5 × 107 PBMC/test where typically 10–20% are CD14+ monocytes. When working with higher cell numbers/number of tests, scale up all volumes accordingly, as shown in Table 1.Wash the BeadsSee Table 1 for volume recommendations.1. Resuspend the beads in the vial (i.e. vortex for >30 sec, or tilt and rotatefor 5 min).2. Transfer the desired volume of beads to a tube.3. Add the same volume of Isolation Buffer, or at least 1 mL, andresuspend.4. Place the tube in a magnet for 1 min and discard the supernatant.5. Remove the tube from the magnet and resuspend the washed beadsin the same volume of Isolation Buffer as the initial volume of beads (step 2).Prepare Cells• Prepare a PBMC suspension according to “General Guidelines”.Resuspend the cells at 1 × 108 cells/mL in Isolation Buffer. • Prepare approximately 10 mL of Isolation Buffer per 5 × 107cells.PBMC: ~2 × 109Figure 1: Purity of human CD3+ cells isolated from PBMC using Dynabeads ® FlowComp ™Human CD3.For support visit /support or *****************************©2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners, except where otherwise stated. LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF .SPEC-06682on labels is the symbol for catalog number.REF Table 1: Volumes for human CD3+ T cells. This protocol is scalable from 1 × 107 to 5 × 108 PBMC. larger tube than recommended in step 14 to successfully remove the biotin in the sample.** When incubating, tilt and rotate the vial so the cells and beads are kept in the bottom of the tube. Do not perform end-over-end mixing if the volume is small relative to the tube size.Description of MaterialsFlowComp ™ Dynabeads ® are uniform, superparamagnetic polystyrene beads (2.8 μm in diameter) coated with modified streptavidin. FlowComp ™ Human CD3 Antibody contains a DSB-X conjugated monoclonal mouse anti-human CD3. FlowComp ™ Release Buffer contains a modified biotin that displaces the modified biotin on the antibody to release cells from the beads.Limited Use Label LicenseThe purchase of this product conveys to the purchaser the limited, nontransferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of itscomponents is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser’s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@ or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.Manufactured by Life Technologies AS, Norway. Life Technologies AS complies with the Quality System Standards ISO 9001:2008 and ISO 13485:2003.Limited Product WarrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies' website at /termsandconditions . If you have any questions, please contact Life Technologies at /support .Isolate CellsThis protocol is based on 5 × 107PBMC, but is directly scalable from 1 × 107to 5 × 108 cells, according to Table 1.1. Transfer 500 μL (5 × 107 cells) prepared cells to a tube and add 25 μLFlowComp ™ Human CD3 antibody. 2. Mix well and incubate for 10 min at 2°C to 8°C.3. Wash by adding 2 mL Isolation Buffer and centrifuge for 8 min at350 × g.4. Remove the supernatant and resuspend in 1 mL Isolation Buffer.5. Add 75 μL washed FlowComp ™ Dynabeads ® and mix well(e.g. vortex 2–3 seconds).6. Incubate for 15 min at room temperature under rolling and tilting.7. Add 1 mL isolation buffer, pipet 2–3 times (or vortex 2–3 seconds) andplace the tube in a magnet for 2 min.8. While the tube is still in the magnet, carefully remove and discard thesupernatant containing the CD3 negative cells.9. Repeat steps 7–8 to wash the bead-bound CD3+ cells. These steps arecritical to obtain a high purity of isolated cells.Release Cells10. Resuspend the bead-bound cells in 1 mL Release Buffer.11. Incubate for 10 min with rolling and tilting at room temperature.12. Pipet 10 times to efficiently release the cells and place in a magnet for2 min. Avoid foaming.13. Transfer the supernatant containing the bead-free CD3+ cells to a newtube, and again place on the magnet for 1 min to remove any residual beads. Transfer again the supernatant containing the bead-free cells to a new tube.14. Add 2 mL Isolation Buffer followed by centrifugation for 8 min at350 × g. Discard the supernatant and resuspend the cell pellet in preferred medium.Keep the cells on 2°C to 8°C until further use in downstream applications.Related Products。
Anti-RFP Affinity Beads 4FF
Anti-RFP Affinity Beads 4FF目录1. 产品介绍 (1)2. 试剂准备 (1)3. 样品纯化 (2)4. 问题及解决方案 (3)5. 订购信息及相关产品 (4)1. 产品介绍红色荧光蛋白（Red Fluorecent Protein，RFP）是从海葵中分离得到的一种分子标记，常用于研究蛋白质的定位、报告基因表达和蛋白-蛋白质相互作用。
Anti-RFP Affinity Beads 4FF 是一种偶联了抗RFP抗体的琼脂糖填料，可以用于天然RFP、RFP突变体及其融合蛋白的免疫沉淀、免疫共沉淀、亲和层析等实验。
本产品具有以下特点：1）使用的抗体和RFP 之间具有极高的亲和力, 可以高效捕获裂解上清中的RFP标签蛋白；2）免疫沉淀后，SDS-PAGE检测，背景更干净，更有利于鉴定蛋白相互作用。
3）琼脂糖基材具有非特异性吸附低，兼容性好的特点，每毫升载量大于1mg(最高可达到5mg/ml RFP结合载量)，可以用于RFP融合蛋白的大量纯化。
此外，我们还提供磁性琼脂糖基材的Anti-RFP Affinity Magarose Beads、高聚物基材的Anti-RFP Affinity Magpoly Beads，可以方便的用于磁性分离。
表1. Anti-RFP Affinity Beads 4FF产品性能指标性能基质高度交联的4%琼脂糖微球配体Anti-RFP Antibody载量>1mg RFP标签蛋白/ml 介质粒径(μm)45-165最大流速0.3 MPa, 3 bar试剂耐受(注意：不影响单链抗体的活性，但是可能影响RFP蛋白的构象) Stable up to 80°C, 1mM DTT, 3M Guanidinium•HCl, 8M Urea, 2M NaCl, 2% Nonidet P40 Substitute, 1% SDS, 1% Triton X-100储存缓冲液20%乙醇储存温度2°C - 8°C2. 试剂准备2.1缓冲液的准备所用水和缓冲液在使用之前建议用0.22μm或0.45 μm滤膜过滤。
分子生物学实验免疫组化实验中抗体稀释液的配制
抗体稀释液的配制Antibody Dilution BuffersPrimary Antibody Dilution Buffer:1%BSA (stabilizer and blocking)0.1% cold fish skin gelatin (blocking)0.05% sodium azide (preservative)0.01M PBS pH7.2 (TBS pH7.6 used in primary antibody dilution buffer produces weaker staining)Note: 1) Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity. 2) This buffer can not be used for diluting HRP conjugated antibodies since sodiu m azide is an inhibitorof HRP.HRP-conjugated Primary Antibody Dilution Buffer:1%BSA (stabilizer)0.1% cold fish skin gelatin (blocking)0.05% Thimerosal (preservative)0.01M PBS pH7.2Note: Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity. Secondary Antibody Dilution Buffer:0.01M PBS, pH 7.20.05% sodium azide (preservative)Note: 1) Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity. 2) Do not use BSA or other serum containing reagents to dilute secondary antibodies since they may bind to BSA or serum therefore reducing antibody affinity. 3) Using TBS to dilute secondary antibodies often produces weaker staining. So use TBS only for the antibodies with high background staining or for alkaline phosphatase conjugated antibodies since phosphate is a inhibitor of alkaline phosphatase.HRP-Streptavidin Dilution Buffer:0.01M PBS, pH 7.20.05% chimerical (preservative)Note: Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity. AP-Streptavidin Dilution Buffer:0.05M TBS, pH 7.60.05% thimerosal (preservative)Note: 1) Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity. 2) PBS can not be used to dilute Streptavidin-AP since phosphate inhibit alkaline phosphatase activity. Fluorescent Dye (Avidin-FITC, Avidin-Texas Red, Avidin-AMCA) Dilution Buffer:0.01M PBS, pH 7.20.05% thimerosal (preservative)。
植物病害诊断试剂盒
植物病害诊断试剂盒美国阿格迪agdia 公司是全球最大的植物病害诊断试剂生产商，产品品种最多，可检测项目多达200多个。
包装规格最全,不同的包装规格适合不同规模的实验室。
从中您一定能发现适合您使用的产品。
选购试剂说明，请仔细阅读。
1，kit, 订货号PSAxxxxx/xxxx 或PSPxxxxx/xxxx 为完整的试剂盒包装，包括样品提取缓冲液、包被好抗体的微孔板（可拆分）、酶标记物、稀释液、缓冲液、底物发色剂、阳性质控（如果应该供应）。
特别注明Indirect ELISA 方法的kit 包括未包被的微孔板及联接用的抗体，所含有的其他组分同上。
2， Reagent Set, 订货号SRAxxxxx/xxxx ,XRAxxxxx/xxxx或SRPxxxxx/xxxx 只含有未包被的微孔板、包被需要的抗体或联接用的抗体、酶标记物。
其他试剂如样品提取缓冲液、稀释液、缓冲液、底物发色剂、质控物需另外订购或自己配制。
我公司销售原厂的上述试剂，详见目录。
3， Bacterial Reagent Set, 订货号BRAxxxxx/xxxx 只含有未包被的微孔板、包被需要的抗体或联接用的抗体、酶标记物。
其他试剂如样品提取缓冲液、稀释液、缓冲液、底物发色剂、质控物需另外订购或自己配制。
我公司销售原厂的上述试剂，详见目录。
4， Bacterial ID订货号BIDxxxxx/xxxx 为完整的试剂盒包装，包括样品提取缓冲液、包被好抗体的微孔板（可拆分）、酶标记物、稀释液、缓冲液、底物发色剂、质控（如果应该供应）。
用于鉴定培养基中或有病症植物提取液中的细菌。
操作简便快速。
5， PS A或SR A中的A代表碱性磷酸酶标记；PS P或SR P中的P代表过氧化物酶标记。
6， Immunostrip test, 为检试纸条，操作简单，几分钟内得到结果，非常适合于现场检测。
该试条必须与相应的样品提取缓冲液配套使用。
实验室需要单独购买该样品提取缓冲液，详见目录。
FLAG M1 Agarose Affinity Gel 产品说明书
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 .。
Protein A G MagBeads 技术手册说明书
Protein A/G MagBeads Cat. No. L00277Technical Manual No. TM0249 Version 08212013 Index1.Product Description2. Instruction For Use3.Troubleshooting4. General Information1.Product Description1.1Intended UseGenScript Protein A/G MagBeads are ideal for small‐scale antibody purification and immunoprecipitation (IP) of proteins, protein complexes or other antigens.1.2PrincipleThe sample containing antibody is added to the Protein A/G MagBeads. The antibody will bind to beads during a short incubation. Then the beads‐bound antibody may be eluted from the beads by using a magnetic separation rack, or used for immunoprecipitation (IP). A cross‐linking procedure may be needed before IP to prevent co‐elution of the primary antibody. Magnetic separation eliminates the changes of micro tubes, minimizes the loss of sample and removes excessive steps of traditional centrifugation method.1.3Description of MaterialMaterial SuppliedGenScript Protein A/G MagBeads are super paramagnetic beads of average 40 μm in diameter, covalently coated with recombinant Protein A/G. The beads are supplied as 25% slurry in phosphate buffered saline (PBS), pH 7.4, containing 20% ethanol. The Protein A/G MagBeads have a binding capacity of more than 10 mg Goat IgG per 1 ml settled beads (e.g. 4 ml 25% slurry).Protein A/G is a genetically engineered protein (MW≈43 kDa) that combines the IgG binding sites of both Protein A and Protein G. 6×His‐tag was attached to its N‐terminal to facilitate the purification. The secreted Protein A/G contains four Fc‐binding domains from Protein A and two from Protein G, making it a more universal tool to bind and purify immunoglobulins.Cat. No. L00277 Size: 2 ml.Additional Material RequiredMixing/Rotation DeviceMagnetic Separation RackTest tubes and pipettesBuffers and solutions (see below)Additional Buffers NeededBinding/Wash Buffer: 20 mM Na2HPO4, 0.15 M NaCl, pH 7.0Elution Buffer: 0.1 M glycine, pH 2‐3Neutralization Buffer: 1 M Tris, pH 8.51×SDS Sample Buffer: 62.5 mM Tris‐HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT,0.01% w/v bromophenol blue2.Instruction For UseThe protocol uses 100 μl Protein A/G MagBeads, this may be scaled up or down accordingly.2.1Preparation of the MagBeadspletely resuspend the beads by shaking or vortexing the vial.2.Transfer 100 μl beads into a clean tube.3.Place the tube on a magnetic separation rack to collect the beads. Remove and discard the supernatant.4.Add 1 ml Binding/Wash Buffer to the tube and invert the tube several times to mix. Use the magnetic separationrack to collect the beads and discard the supernatant. Repeat this step twice.2.2Separation of Target IgG1.Resuspend the beads in 100 μl Binding/Wash Buffer.2.Add the sample containing target IgG to the tube and gently invert the tube to mix.3.Incubate the tube at room temperature with mixing (on a shaker or rotator) for 30 – 60 minutes.e the magnetic separation rack to collect the beads and discard the supernatant. If necessary, keep thesupernatant for analysis.5.Add 1 ml Binding/Wash Buffer to the tube and mix well, use the magnetic separation rack to collect the beads anddiscard the supernatant. Repeat the wash step three more times.6.Proceed to elution of isolated IgG (Section 2.3).2.3Elution of Isolated IgG1.Add 100 μl Elution Buffer to the tube and mix well. Incubate for five minutes at room temperature with occasionalmixing.e the magnetic separation rack to collect the beads and transfer the supernatant that contains the eluted IgG intoa clean tube.3.Repeat Step 1 and 2 twice.4.Add 10 μl of Neutralization Buffer to each 100 μl eluate to neutralize the pH. If needed, perform a buffer exchangeby dialysis or desalting.2.4ImmunoprecipitationBound IgG will be co‐eluted along with the target when using elution methods. If the presence of IgG does not disturb desired detection system, go directly to section 2.4.2 below. For applications where co‐elution of the IgG is not desired, the primary IgG can be cross‐linked to the Protein A/G MagBeads as described in section 2.4.1 below.2.4.1Cross‐linking IgG to the Beads1.Add 1 ml 0.2 M triethanolamine, pH 8.2 to the Protein A/G MagBeads with immobilised IgG. Wash twice using themagnetic separation rack with 0.2 M triethanolamine, pH 8.2 as the washing buffer.2.Resuspend the beads in 1 ml of 20 mM dimetyl pimelimidate dihydrochloride (DMP) in 0.2 M triethanolamine, pH 8.2(5.4 mg DMP/ml buffer). This cross‐linking solution must be prepared freshly.3.Incubate the beads with rotational mixing for 30 minutes at room temperature. Use the magnetic separation rack tocollect the beads and discard the supernatant.4.Resuspend the beads in 1 ml of 50 mM Tris, pH 7.5 to stop the reaction and incubate for 15 minutes at roomtemperature with rotational mixing.e the magnetic separation rack to collect the beads and discard the supernatant.6.Wash the cross‐linked beads three times with 1 ml PBS, pH7.4.2.4.2Binding Antigen to the IgG Cross‐linked Beads1.Add sample containing target antigen to the beads. For a 100 kD protein, use a volume containing approximate 25 µgtarget antigen/ml beads to assure an excess of antigen. If dilution of antigen is necessary, PBS or 0.1 M phosphate buffer (pH 7‐8) can be used as dilution buffer.2.Incubate with tilting and rotation for one hour at room temperature.3.Place the tube on the magnetic separation rack for 2 minutes to collect the IgG‐coated Beads‐target complex. Forviscous samples, double the time on the rack. Discard the supernatant.4.Wash the beads 3 times using 1 ml PBS.2.4.3Elution of Target ProteinA.Denaturing elution1.Place the tube from section2.4.2 on the magnetic separation rack to collect the beads and discard the supernatant.2.Add 100 µl 1XSDS Sample Buffer to the tube and mix well.3.Heat the tube at 100°C for five minutes.e the magnetic separation rack to collect the beads and transfer the supernatant containing desired sample into anew tube.5.Analyze the sample by SDS‐PAGE followed by Western blot analysis.B.Non‐denaturing elution1.Place the tube from section2.4.2 on the magnetic separation rack to collect the beads and discard the supernatant.2.Add 100 µl Elution Buffer to the tube and mix well. Incubate for five minutes at room temperature with occasionalmixing.e the magnetic separation rack to collect the beads and transfer the supernatant into a new tube.4.Repeat Step 2 and 3 twice.5.Add 10 μl Neutralization Buffer to each 100 μl of eluate to neutralize the pH.3.TroubleshootingReview the information below to troubleshoot your experiments using the GenScript Protein A/G MagBeads. Problem Possible Cause SolutionThe beads are difficult toimmobilize using the magneticseparation rack.Too many beads are used.Decrease the volume of MagBeadssuspension.A considerable amount of samplehas been added, but very fewspecific antibody of interest isdetected.The antibody of interest is at verylow concentration.Use a serum‐free medium for cellsupernatant samples.Affinity‐purify the antibody using itsspecific antigen coupled to anaffinity supporting material.The antibody of interest is purified,but it is degraded (as determined byloss of function in downstreamassay).The antibody is sensitive to low‐pHelution buffer.The downstream application issensitive to the neutralized elutionbuffer.Try another elution reagent, such as3.5 M MgCl2, 10 mM phosphate, pH7.2.Desalt or dialyze the eluted sampleinto a suitable buffer.No antibody is detected in anyeluate.The antibody in the sample cannotbind to Protein A/G.Try GenScript Protein A MagBeadsor Protein G MagBeads.4.General Information4.1Storage and StabilityThis product is stable until the expiration date stated on the COA, when stored unopened at 2–8°C. Do not freeze the product. Keep the MagBeads in liquid suspension during storage and all handling steps. Drying will cause loss of binding capacity and result in reduced performance. Resuspend the beads well before use. Be careful to avoid bacterial/fungal contamination.4.2Technical SupportPlease contact GenScript for further technical information (see contact details). Certificate of Analysis/Compliance is available upon request. The latest revision of the package insert/instructions for use is available on .4.3Warning and LimitationsThis product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. This product contains 20 % EtOH as a preservative. Flammable liquid and vapor. Flash point 38°C. R‐10 flammable. Material Safety Data Sheet (MSDS) is available at .4.4Related MagBeads ProductsCat. No. Product NameL00273 Protein A MagBeadsL00274 Protein G MagBeadsL00295 Ni‐Charged MagBeadsL00327 Glutathione MagBeadsL00275 Mouse Anti‐His mAb MagBeadsL00336 Mouse Anti‐GST mAb MagBeadsGenScript USA Inc.860 Centennial Ave.,Piscataway, NJ 08854Toll‐Free: 1‐877‐436‐7274Tel: 1‐732‐885‐9188, Fax: 1‐732‐210‐0262Email: *********************Web: 。
Anti-FLAG（DYKDDDDK）免疫磁珠说明书
Anti-FLAG（DYKDDDDK）免疫磁珠Cat#：MIP0064（本品仅用于科学研究，不可用于诊断及治疗）一．背景信息Flag-Tag（DYKDDDDK），常用于真核蛋白质重组表达标记。
Anti-FLAG（DYKDDDDK）免疫磁珠，由高品质的FLAG抗体与磁珠共价偶联制成, 具有高载量（至少为0.6mg protein/ml磁珠），高特异性，操作迅速便捷，性质稳定，可反复使用的特点，可用于FLAG标签融合蛋白的亲和纯化及免疫（共）沉淀。
二．性能指标应用范围：可用于Met修饰的N端FLAG融合蛋白（Met-FLAG–Protein），N端FLAG融合蛋白（FLAG–Protein）和C端FLAG融合蛋白（Protein-FLAG）的亲和纯化及免疫（共）沉淀。
抗体属性：小鼠单克隆抗体，IgG2a亚型。
磁珠属性：琼脂糖包裹的超顺磁珠，平均粒径50um。
载量：0.5mL纳米磁珠，共价偶联2mg Anti-FLAG单克隆抗体，可纯化或沉淀至少0.6mg FLAG融合蛋白。
强度：竞争洗脱，可反复使用5次以上。
成分：0.5mL共价偶联Anti-FLAG单克隆抗体的磁珠, 溶于1ml PBS中。
保存方法：在加入了0.2‰叠氮钠的PBS保存液中，4℃可保存1年。
三．说明书阅读指南四．用前必读1.以下试剂及设备，需要客户自备，试剂推荐配方见说明书附件1, 推荐蛋白质制备方案见附件2。
磁力架1个，1.5mL 离心管若干细胞裂解液，酸性洗脱液，中和液，PBS，2x蛋白上样缓冲液, 3xFLAG多肽(货号Q7007)待测抗原样品及检测抗体2.注意事项（开箱前必读）1)本产品在在加入了0.2‰叠氮钠的PBS保存液中，4℃可保存1年，冷藏条件下运输。
2)勿离心、冷冻、干燥磁珠，勿使用超声处理磁珠，勿使酸处理磁珠时间超过10min。
3)混匀磁珠时，请采用移液枪轻柔吹打，柔和涡旋，上下颠倒及摇床混匀等方法。
勿使用超声等方法。
免疫共沉淀中的缩写
免疫共沉淀中的缩写引言免疫共沉淀（Immunoprecipitation）是一种常用的实验室技术，用于研究蛋白质的相互作用和功能。
在免疫共沉淀实验中，研究人员利用抗体的特异性与目标蛋白质结合，然后通过沉淀过程将蛋白质从细胞或组织中分离出来。
该技术通常与其他分析方法（如免疫印迹、质谱分析等）结合使用，以进一步研究蛋白质间的相互作用以及其在细胞过程中的功能。
在进行免疫共沉淀实验时，研究人员通常会使用缩写来简化术语的表达，提高实验效率。
本文将介绍一些常见的免疫共沉淀中的缩写，其中包括实验所需的材料、抗体以及相关方法。
免疫共沉淀实验中的常见缩写1. 免疫共沉淀材料•IP Buffer（Immunoprecipitation Buffer）：免疫共沉淀缓冲液，用于裂解细胞并维持蛋白质的稳定性。
•Lysis Buffer：裂解缓冲液，也用于裂解细胞并提取蛋白质，与IP Buffer 类似。
•RIPA Buffer（Radioimmunoprecipitation Assay Buffer）：放射免疫共沉淀试剂盒缓冲液，用于裂解细胞并增强蛋白质的溶解性。
•Protein A/G Beads（Protein A/G Sepharose Beads）：蛋白 A/G 球，用于与抗体结合，从而富集目标蛋白质。
•PMSF（Phenylmethylsulfonyl Fluoride）：苯甲基磺酰氟，用作蛋白酶抑制剂，防止蛋白质在提取过程中被降解。
•BSA（Bovine Serum Albumin）：牛血清白蛋白，用作抗体稀释液的成分。
2. 免疫共沉淀抗体•IP Antibody：免疫共沉淀抗体，用于与目标蛋白质特异性结合，富集目标蛋白质。
•Primary Antibody：一抗，与目标蛋白质结合并形成免疫复合物。
•Secondary Antibody：二抗，与一抗结合并标记荧光染料或酶，便于免疫复合物的检测。
•HRP（Horseradish Peroxidase）：辣根过氧化物酶，常用于标记二抗或直接标记抗体，通过触发荧光或发色反应来检测免疫共沉淀结果。
protein A磁珠免疫沉淀DynabeadsProteinA_man
• For sensitive proteins and phosphorylation studies, the isolation protocol
Catalog nos. 10001D, 10002D, 10008D
Store at 2˚C to 8˚C
including elution may be performed at 4°C, to avoid protein complex
Product Description
Dynabeads® Protein A are designed for immunoprecipitation of proteins, protein complexes, protein-nucleic acid complexes, and other antigens.
Figure 1: Principle of immunoprecipitation of antigen using Dynabeads® Protein A.
Required Materials
• Magnet (DynaMag™). See /magnets for recommendations.
Rev. Date: October 2011 (Rev. 007)
dissociation and minimize enzymatic a
Cat. no. 10001D 10002D 10008D
Volume 1 mL 5 mL 50 mL
Dynabeads® Protein A contains 30 mg Dynabeads®/mL in phosphate buffered saline (PBS), pH 7.4, with 0.01% Tween®-20 and 0.09% sodium azide as a preservative.
常见蛋白质标签
常见蛋白质标签总结2008-12-08 22:06Protein tags are peptide sequences genetically grafted onto a recombinant protein. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing. Tags are attached to proteins for various purposes.一、氨基酸标签（含小肽标签）A stretch of amino acids is added to the protein and enables the recovery of the labelled protein by its unique affinity. Usually its easiest to add the tag to either end of the protein to ensure its accessibility and not to disturb the protein folding.组氨酸标签（His tag）一般为6个组氨酸，用Ni2+ （Cu2+）亲和层析纯化FLAG tag ：N-DYKDDDDK-C ，recovered with specific antibodyHA tag：an epitope derived from the Influenza protein haemagglutinin (HA，禽流感病毒血凝素)，e.g. N-YPYDVP-C，recovery with an HA antibodyMYC tag：an epitope derived from the human proto-oncoprotein MYC，e.g.N-ILKKATAYIL-C, N-EQKLISEEDL-C，recovery with an MYC antibodySBP tag：Streptavidin Binding Peptide，链霉亲合素结合肽，38 amino acid tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)，更多参考在SigmaCBP tag：钙调蛋白结合肽（CBP; 26aa）钙调蛋白结合肽与钙调素结合是Ca2+依赖的，这种结合不受标签所处的位置影响（N端和C端均可），在中性pH条件下使用2mM EGTA可以很方便的将目标蛋白洗脱下来。
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Anti-DYKDDDDK Affinity Beads
目录
1. 产品介绍 (1)
2. 试剂准备 (1)
3. 样品纯化 (2)
4．试剂兼容性 (3)
5. 问题及解决方案 (4)
6. 订购信息及相关产品 (4)
1. 产品介绍
Flag标签是一个由八个亲水氨基酸组成的多肽片段，定位在融合蛋白表面，因此更易与抗体结合以及被肠激酶分解。
Anti-DYKDDDDK Affinity Beads是以抗Flag(DYKDDDDK)抗体为亲和配体，一步纯化原核、酵母或哺乳动物细胞表达的Flag标签融合蛋白。
Anti-DYKDDDDK Affinity Beads以4%琼脂糖凝胶为基质，杂蛋白非特异性结合少，可用于Flag标签融合蛋白的纯化和免疫沉淀（IP）。
表1. Anti-DYKDDDDK Affinity Beads产品性能
指标性能
基质4%琼脂糖微球
配体Anti-DYKDDDDK鼠单克隆抗体
载量>1mg DYKDDDDK标签蛋白/ml 介质
粒径(μm)45-165
最大流速0.1 MPa, 1 bar
储存缓冲液0.02%叠氮化钠，1XPBS
储存温度2°C - 8°C
2. 试剂准备
2.1缓冲液的准备
所用水和缓冲液在使用之前建议用0.22μm或0.45 μm滤膜过滤。
平衡/洗杂液: 50mM Tris，0.15M NaCl，pH7.4
洗脱液: 0.1M glycine HCl，pH3.0
竞争性洗脱液：50mM Tris，0.15M NaCl, 100-300µg Flag多肽/ml，pH7.4
中和液：1M Tris-HCl，pH8.5
2.2 样品准备
上柱之前要确保样品溶液有合适的离子强度和pH值，可以用平衡液对样品或细胞培养液稀释，或者用平衡液透析。
样品在上样前建议离心或用0.22μm或0.45μm滤膜过滤，减少杂质，提高蛋白纯化效率和防止堵塞柱子。
3. 样品纯化
3.1 柱层析
1) 将Anti-DYKDDDDK Affinity Beads装入合适的层析柱，层析用5 倍柱体积的平衡液进行平衡，使填料处于与目的蛋白相同的缓冲体系下。
2) 将样品加到平衡好的Anti-DYKDDDDK Affinity Beads中，收集流出液，可以反复上样增加结合效率。
3) 用10倍柱体积的洗杂液进行清洗，去除非特异性吸附的杂蛋白，收集洗杂液。
4) A酸性洗脱：使用5倍柱体积的洗脱液洗脱，收集管中预先加好中和液，加入量15-25µl 中和液/ml洗脱液，分管收集。
注：酸性洗脱后填料要立即用平衡液平衡，Anti-DYKDDDDK Affinity Beads在洗脱液中不要超过20min。
B竞争性洗脱：使用5倍柱体积的竞争性洗脱液洗脱。
分管收集。
5) 使用3倍柱体积的洗脱液再生，然后用平衡液平衡至中性。
6) 然后保存在含0.02%叠氮化钠的PBS溶液中，2-8度保存。
3.2 静态吸附
1) 填料准备：取适量的Anti-DYKDDDDK Affinity Beads加入层析柱中，流干保护液。
加入5倍柱体积的平衡液清洗。
2) 加入样品溶液，4度或室温震荡孵育至少30min(不能磁力搅拌)，确保填料与样品溶液充分混合。
3) 孵育完毕后，将填料混合液离心（1000x g离心5min）或过滤收集填料。
4) 将填料装入层析柱中，用平衡液清洗直至紫外稳定。
5) 用酸性洗脱液或竞争性洗脱液洗脱，参考3.1 中4）。
6) 填料再生和保存参考3.1中5)和6)。
3.3 免疫共沉淀操作流程
1) 填料准备：取40µl的Anti-DYKDDDDK Affinity Beads (柱体积20µl)混合液加入到2ml 离心管中，5000Xg离心30秒，吸弃上清。
2) 填料加入0.5ml平衡液，悬浮填料（使填料处于与目的蛋白相同的缓冲体系下，起到保护蛋白的作用），5000Xg离心30秒，吸弃上清。
重复一次。
3) 加入200-1000µl样品裂解液到处理好的填料中，混合均匀，在室温下置于翻转混合仪轻轻翻转离心管，促使样品和填料充分接触并吸附，室温至少1小时。
5000Xg离心30秒，吸弃上清。
4) 洗杂：加入0.5ml的洗杂液，悬浮填料，进行清洗，去除非特异性吸附的杂蛋白，5000Xg
离心30秒，吸弃上清。
再重复三次。
确保去除非特异性吸附。
5) 样品洗脱：可根据后期检测的需要选择不同的洗脱方法。
A:酸性洗脱
加入100µl洗脱液，悬浮填料。
室温孵育5min, 5000Xg离心30秒。
小心取出上清，不要吸到填料，用中和液中和。
洗脱样品放置4度，长时间放置-20度保存。
B:竞争性洗脱
加入100µl竞争性洗脱液洗脱。
室温孵育30min, 5000Xg离心30秒。
小心取出上清，不要吸到填料。
洗脱样品放置4度，长时间放置-20度保存。
C:变性洗脱
样品缓冲液中含有β-巯基乙醇和DTT,可以使填料中抗体重链和轻链断开。
含有SDS的样品缓冲液可以使Anti-DYKDDDDK抗体变性，洗脱后的Anti-DYKDDDDK Affinity Beads没办法重复使用。
每管中加入20µl 2X Loadding Buffer，95℃加热5min。
5000Xg离心30秒，吸取上清SDS-PAGE电泳检测。
4．试剂兼容性
表2、Anti-DYKDDDDK Affinity Beads试剂兼容性
试剂名称最大耐受浓度备注
β-巯基乙醇10mM DTT 80mM SDS -- 纯化过程中应避免使用，如果在IP中使用，填料不能回收重复利用。
EDTA 5mM 过高的EDTA会降低蛋白回收率。
Tween-20 5%
Triton X-100 5%
NP-40 4%
过高浓度会影响标签蛋白结合效率。
盐酸胍0.3M
尿素 1.5M
过高浓度会使抗体变性。
甘油20% 过高浓度会影响标签蛋白结合。
氯化钠1M 减少非特异性吸附
5. 问题及解决方案
问题
原因分析 推荐解决方案
填料过载 减少上样体积或增加填料体积。
结合时间太短 延长样品和填料的结合时间。
标签未暴露 可以加入低浓度的变性剂，上样前透析。
流穿中有目的蛋白
溶液中试剂不兼容
样品上样前进行透析。
目的蛋白不稳定
使用新配制的样品。
低温操作。
细胞裂解液中加入蛋白酶抑制剂。
样品中无标签融合蛋白 纯化前Western 检测是否有Flag 标签 洗脱组分中没有目的蛋白
目的蛋白表达量太低 优化蛋白表达量。
增加上样量。
减少NaCl 浓度。
非特异性吸附 减少上样量
背景太杂
洗杂不充分
增加洗杂次数，每次清洗孵育5-10min 。
增加洗杂液中盐离子浓度。
6. 订购信息及相关产品
名称
货号 规格 SA042001 1ml Anti-DYKDDDDK Affinity
Beads
SA042005 5ml SA068001 1ml Anti-HA Affinity Beads SA068005 5ml SA070001 1ml Anti-GFP Affinity Beads 4FF SA070005 5ml SA065001 1ml Anti-c-Myc Affinity Beads SA065005 5ml SA072001 1ml Anti-RFP Affinity Beads 4FF SA072005
5ml。