FLAG标签融合蛋白纯化试剂盒使用说明书
碧云天生物技术pCMV-N-Flag产品说明书
pCMV-N-Flag产品编号 产品名称 包装 D2722-1µg pCMV-N-Flag 1µg D2722-100µgpCMV-N-Flag100µg产品简介：pCMV-N-Flag 是碧云天自行研发的用于在哺乳动物细胞中表达N 端和Flag tag (Flag 标签)融合的目的蛋白的表达质粒。
含有CMV 启动子可以高效启动目的蛋白在细胞中的表达；在多克隆位点的5'端含有一个可以编码Flag 标签的序列，因此可以表达出含有Flag 标签的融合蛋白，可以方便地使用抗Flag 的抗体来识别目的蛋白，有利于目的蛋白检测和分离纯化。
质粒为卡那霉素抗性。
转染细胞后，可使用G418筛选稳定表达目的蛋白的细胞株。
pCMV-N-Flag 质粒的主要信息如下：Feature Nucleotide Position CMV promoter 1–602 T3 promoter and T3 primer binding site 620–639 Flag tag 679–702 multiple cloning site 705–778 T7 promoter and T7 primer binding site 821–842 SV40 polyA signal 854–1237 f1 origin of ss-DNA replication 1375–1681 bla promoter 1706–1830 SV40 promoter 1850–2188 neomycin/kanamycin resistance ORF 2223–3014 HSV-thymidine kinase (TK) polyA signal 3015–3473 pUC origin 3602–4269 pCMV-N-Flag 质粒的图谱如下：碧云天生物技术/Beyotime Biotechnology 订货热线: 400-1683301或800-8283301 订货e-mail :******************技术咨询: *****************网址: 碧云天网站 微信公众号pCMV-N-Flag的多克隆位点的详细图谱如下：Flag tag __SacI M D Y K D D D D K651 GAGCTCCACC GCGGTGGCGG CCGCCATGGA TTACAAGGAT GACGACGATACTCGAGGTGG CGCCACCGCC GGCGGTACCT AATGTTCCTA CTGCTGCTATXmaI PstISmaI BamHI HindIII EcoRI EcoRV SalI BglII701 AGAGCCCGGG CGGATCCAAG CTTCTGCAGG AATTCGATAT CGTCGACAGATCTCGGGCCC GCCTAGGTTC GAAGACGTCC TTAAGCTATA GCAGCTGTCTXhoI XbaI ApaI751 TCTCTCGAGT CTAGAACTAG TGGGCCCGGT ACCTTAATTA ATTAAGGTACAGAGAGCTCA GATCTTGATC ACCCGGGCCA TGGAATTAAT TAATTCCATGpCMV-N-Flag中没有的酶切位点(Restriction enzymes that do not cut pCMV-N-Flag)包括：Afl II Age I Ahd I Asc I Bbs I Bbv II Blp I Bsg I BsiW I BsmB I BspM II BsrG I BssH II Bst1107 I BstE II Ear I Eco47 III Eco72 I EcoN I Esp I Fse I Nru I PflM I Pme I Pml I PpuM I Psp1406 I Sap I Sca I Spl IpCMV-N-Flag中的单酶切位点(Restriction enzymes that cut pCMV-N-Flag once)包括：Nde I CA`TA,TG 241 SnaB I TAC|GTA 347 Nhe I G`CTAG,C 598 Sac I G,AGCT`C 656 Sac II CC,GC`GG 663 BstX I CCAN,NNNN`NTGG 664 Not I GC`GGCC,GC 669 PspA I C`CCGG,G 706 Xma I C`CCGG,G 706 Srf I GCCC|GGGC 708 Sma I CCC|GGG 708 BamH I G`GATC,C 713 Hind III A`AGCT,T 719 Pst I C,TGCA`G 729 EcoR I G`AATT,C 731 EcoR V GAT|ATC 739 Sal I G`TCGA,C 743 Acc I GT`MK,AC 744 Bgl II A`GATC,T 749 PaeR7 I C`TCGA,G 755 Xho I C`TCGA,G 755 Xba I T`CTAG,A 761 Spe I A`CTAG,T 767 Bsp120 I G`GGCC,C 773 Apa I G,GGCC`C 777 Pvu I CG,AT`CG 855 Bcl I T`GATC,A 1009 Mun I C`AATT,G 1102 Hpa I GTT|AAC 1115 Mlu I A`CGCG,T 1238 Dra III CAC,NNN`GTG 1468 Sfi I GGCCN,NNN`NGGCC 2127 BseR I GAGGAG 16/14 2170 Stu I AGG|CCT 2173 Cla I AT`CG,AT 2192 Kas I G`GCGC,C 2351 Nar I GG`CG,CC 2352 Ehe I GGC|GCC 2353 Bbe I G,GCGC`C 2355 Msc I TGG|CCA 2434 Tth111 I GACN`N,NGTC 2470 BsrD I GCAATG, 8 2585 Bsp1286 I G,DGCH`C 2655 Rsr II CG`GWC,CG 2868 BsiC I TT`CG,AA 3034 BstB I TT`CG,AA 3034 Bsa I GGTCTC 7/11 3341 HgiE II ACCNNNNNNGGT-1/13 3681 ApaL I G`TGCA,C 3956pCMV-N-Flag质粒中对于插入片段进行测序时，推荐使用的正向测序引物T3和反向测序引物T7的序列如下：T3 primer (620–639): 5' AATTAACCCTCACTAAAGGG 3'T7 primer (821-842): 5' GTAATACGACTCACTATAGGGC 3'pCMV-N-Flag的全序列信息请参考碧云天网站上该质粒的信息。
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。
【免费下载】蛋白纯化试剂盒说明书
变性剂 洗涤剂 其他添加剂
缓冲液
10 mmol/L 还原型谷胱甘肽
8 mol/L尿素 6 mol/L 盐酸胍
2% TritonX-100(非离子物质) 2% Tween 20（非离子物质） 2% NP-40 (非离子物质) 2% 胆酸盐 1% CHAPS（两性离子）
20% 乙醇 50% 甘油 100 mmol/L Na2SO4 1.5 mol/L NaCl 1 mmol/L EDTA 60 mmol/L柠檬酸盐 100 mmol/L 磷酸钠，pH 7.4
洗脱液 唑，pH 8.0
20 mmol/L Tris-HCl，8 mol/L 尿素，500 mmol/L NaCl，500 mmol/L 咪
注意：样品和缓冲液中适当浓度的咪唑对获得最大的纯化是必需的，而且产量因蛋白 而不尽相同。自然条件下，20-40 mmol/L 的咪唑的几盒缓冲液对许多蛋白已经足够。洗脱 液中500 mmol/L 咪唑绝大多数情况下完全可洗下目的蛋白。
含螯合剂，例如EDTA，EGTA，柠檬酸等
20%乙醇
4或30℃
5 mmol/L DTE(二硫赤藓糖醇) 5 mmol/L DTT(二硫苏糖醇)
20 mmol/L β-巯基乙醇 5 mmol/L TCEP(磷酸三氯乙酯)
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FLAG Peptide F3290 产品说明书
F3290 Rev 02/221Product InformationFLAG ® PeptideF3290Storage Temperature 2-8 °CProduct DescriptionMolecular Weight: 1013.0Sequence: N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C (synthetic octapeptide)The FLAG ® Peptide is useful for the competitive elution of amino-terminal, Met-amino-terminal or carboxy-terminal FLAG ® fusion proteins from the ANTI-FLAG ® M1 or M2 monoclonal antibody in solution or bound to agarose gel. A workingconcentration of 100 µg/mL is commonly used toelute FLAG ® fusion proteins from the ANTI-FLAG ® M1 and M2 affinity resins.2-4 Five one-column volumes of the working solution are sufficient to elute most FLAG ® fusion proteins.Various publications have used the FLAG ® Peptide (Cat. No. F3290) at other concentrations, which we have not necessarily tested, such as: • 150 µg/mL 5 • 200 µg/mL 6 • 250 µg/mL 7 • 300 µg/mL 8,9•0.5 mg/mL (500 µg/mL)10The FLAG ® Peptide will not elute 3X FLAG ® fusion proteins. For this application, use the 3X FLAG ® Peptide (Cat. No. F4799).Precautions and DisclaimerFor research use only. Not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.Preparation InstructionsTo prepare a stock solution, dissolve in TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) to a finalconcentration of 5 mg/mL. Aliquot and store at −20 °C. Repeated freezing and thawing is not recommended.Storage/StabilityStore the lyophilized powder at 2-8 °C. Afterreconstitution, aliquot and store at −20 °C. Repeated freezing and thawing is not recommended.ProcedureElution of FLAG ® Fusion Proteins by Competition with FLAG ® Peptide: Elute the bound FLAG ® fusion protein from the ANTI-FLAG ® M1 or M2 affinity resin bycompetitive elution with five one-column volumes of a solution containing 100 µg/mL FLAG ® peptide in TBS.References1. Brizzard, B.L. et al ., BioTechniques , 16(4):730-735 (1994). 2. Antonysamy, S. et al ., Proc. Nat. Acad. Sci. USA ,109(44): 17960-17965 (2012). 3. He, J. et al ., Nucleic Acids Res ., 40(13):6097-6108 (2012). 4. Singh, O. et al ., Protein Sci ., 22(8):1071-1077 (2013). 5. Li, W. et al ., Mol. Cell. Biol ., 36(22):2855-2866 (2016). 6. Cheng, F. et al ., Mol. Immunol ., 60(1):44-53 (2014). 7. Singh, G. et al ., Methods , 65(3):320-332 (2014). 8. Meller, N. et al ., J. Biol. Chem ., 279(36):37470-37476 (2004). 9. Aguado, L.C. et al ., Nature , 547(7661):114-117 (2017). 10. Simsek, D. et al ., Cell , 169(6):1051-1065.18 (2017).The life science business of Merck operatesas MilliporeSigma in the U.S. and Canada.Merck and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates.All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.F3290 Rev 02/22 2NoticeWe 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 our customers 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 a commitment 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.。
GFP标签融合蛋白亲和凝胶使用说明书
Anti-GFP亲和凝胶Cat#：IP0057（本品仅用于科学研究，不可用于诊断及治疗）1.背景信息GFP，绿色荧光蛋白(Green fluorescent protein)，来源于维多利亚多管发光水母，由约238个氨基酸组成，从蓝光到紫外线都能使其激发出绿色萤光。
GFP常用于真核蛋白质重组表达标记。
Anti -GFP亲和凝胶，由高品质的GFP鼠单克隆抗体与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GFP标签融合蛋白的免疫（共）沉淀检测。
2.性能指标应用范围：可用于N端GFP融合蛋白（GFP–Protein）和C端GFP融合蛋白（Protein-GFP）的免疫（共）沉淀。
载量：1ml Sepharose 4B琼脂糖颗粒，共价偶联800μg Anti-GFP 鼠单克隆抗体，可沉淀至少1.2mg GFP融合蛋白。
成分：共价偶联Anti-GFP 鼠单克隆抗体的Sepharose 4B琼脂糖颗粒, 1ml溶于2ml PBS+50%甘油中。
保存方法：在加入了50%甘油和0.2‰叠氮钠的PBS保存液中，-20℃可保存1年。
3.使用方法3.1细胞裂解液制备3.1.1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。
3.1.2预冷的PBS(pH7.2)重悬细胞，1000rpm离心3min，弃上清。
重复一次。
3.1.3根据细胞的量加入相应体积的细胞裂解液（推荐配方见5.3），反复吹打后冰上放置10-20min，让细胞充分裂解。
3.1.4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。
冰上放置30min之后，12000rpm，4℃离心10min。
取上清，冷冻保存。
3.2装柱及孵育3.2.1根据使用目的选用重力柱或离心管，移入适量亲和凝胶。
FLAG tag Peptide_多功能融合标签。_98849-88-8_Apexbio
产品名: FLAG tag Peptide 修订日期: 6/30/2016产品说明书化学性质产品名:FLAG tag Peptide Cas No.:98849-88-8 分子量:1012.97 分子式:C41H60N10O20 别名:H-Asp-Tyr-Lys-Asp-Asp-Asp-As p-Lys-OH 化学名:FLAG Peptide SMILES: C1=CC(=CC=C1CC(C(=O)NC(CCCCN)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CCCCN)C(=O)O)NC(=O)C(CC(=O)O)N)O溶解性: >50.6mg/mL in DMSO储存条件: Desiccate at -20°C一般建议: For obtaining a higher solubility , please warm the tube at 37°Cand shake it in the ultrasonic bath for a while.Stock solution can bestored below -20°C for several months.运输条件:Evaluation sample solution : ship with blue iceAll other available size: ship with RT , or blue ice upon request生物活性靶点 :Tag Peptides 信号通路:产品描述:FLAG 标签肽（FLAG tag Peptide ）是一个含有肠激酶酶切位点的8肽（Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys ），专门用于免疫亲和层析。
FLAG 标签肽融合系统主要用于蛋白鉴定与纯化。
蛋白纯化试剂盒说明书
蛋白纯化试剂盒介绍试剂盒有10个重力流柱，而该柱在自然条件下纯化可重新装填Ni-IDA Sefinose TM 6 Fast Flow 和结合缓冲液，洗脱液。
这10个柱子通过固定化金属亲和层析可用于纯化带组氨酸标签的蛋白。
Ni-IDA Sefinose 6 Fast Flow 有很高的蛋白结合能力，低镍离子（Ni2 +）的泄漏以及可与变性剂和广泛的添加剂相兼容。
澄清和非澄清的样品可使用该柱。
特殊的frits 在纯化过程可防止介质变干燥。
一次纯化平均需要30分钟。
在变性下纯化，请自备额外的结合缓冲液和洗脱液。
特征柱材料聚丙烯桶，聚乙烯筛板介质Ni-IDA Sefinose 6 Fast Flow平均粒径90 µm蛋白结合能力约6 mg组氨酸标签蛋白/1毫升树脂床体积 1 mL/柱使用时兼容性在各种通用缓冲液，洗涤剂和变性剂中稳定避免在缓冲液中含螯合剂，例如EDTA，EGTA，柠檬酸等pH稳定性，短期（2h）2-14储存20%乙醇保存温度4或30℃与NI-IDA树脂相兼容的试剂还原试剂5 mmol/L DTE(二硫赤藓糖醇)5 mmol/L DTT(二硫苏糖醇)20 mmol/L β-巯基乙醇5 mmol/L TCEP(磷酸三氯乙酯)10 mmol/L 还原型谷胱甘肽变性剂8 mol/L尿素6 mol/L 盐酸胍洗涤剂2% TritonX-100(非离子物质)2% Tween 20（非离子物质）2% NP-40 (非离子物质)2% 胆酸盐1% CHAPS（两性离子）其他添加剂20% 乙醇50% 甘油100 mmol/L Na2SO41.5 mol/L NaCl1 mmol/L EDTA60 mmol/L柠檬酸盐100 mmol/L 磷酸钠，pH 7.4 缓冲液100 mmol/L Tris-HCl pH 7.4100 mmol/L Tris pH 7.4100 mmol/L HEPES pH 7.4100 mmol/L MOPS pH 7.4100 mmol/L 醋酸钠pH 4注意：1.为了最佳的操作过程，按照上述纯化步骤进行预洗以除去任何削弱结合镍离子的物质。
蛋白纯化试剂盒说明书
蛋白纯化试剂盒介绍试剂盒有10个重力流柱，而该柱在自然条件下纯化可重新装填Ni-IDA Sefinose TM 6 Fast Flow 和结合缓冲液，洗脱液。
这10个柱子通过固定化金属亲和层析可用于纯化带组氨酸标签的蛋白。
Ni-IDA Sefinose 6 Fast Flow 有很高的蛋白结合能力，低镍离子（Ni2 +）的泄漏以及可与变性剂和广泛的添加剂相兼容。
澄清和非澄清的样品可使用该柱。
特殊的frits 在纯化过程可防止介质变干燥。
一次纯化平均需要30分钟。
在变性下纯化，请自备额外的结合缓冲液和洗脱液。
特征柱材料聚丙烯桶，聚乙烯筛板介质Ni-IDA Sefinose 6 Fast Flow平均粒径90 µm蛋白结合能力约6 mg组氨酸标签蛋白/1毫升树脂床体积 1 mL/柱使用时兼容性在各种通用缓冲液，洗涤剂和变性剂中稳定避免在缓冲液中含螯合剂，例如EDTA，EGTA，柠檬酸等pH稳定性，短期（2h）2-14储存20%乙醇保存温度4或30℃与NI-IDA树脂相兼容的试剂还原试剂5 mmol/L DTE(二硫赤藓糖醇)5 mmol/L DTT(二硫苏糖醇)20 mmol/L β-巯基乙醇5 mmol/L TCEP(磷酸三氯乙酯)10 mmol/L 还原型谷胱甘肽变性剂8 mol/L尿素6 mol/L 盐酸胍洗涤剂2% TritonX-100(非离子物质)2% Tween 20（非离子物质）2% NP-40 (非离子物质)2% 胆酸盐1% CHAPS（两性离子）其他添加剂20% 乙醇50% 甘油100 mmol/L Na2SO41.5 mol/L NaCl1 mmol/L EDTA60 mmol/L柠檬酸盐100 mmol/L 磷酸钠，pH 7.4 缓冲液100 mmol/L Tris-HCl pH 7.4100 mmol/L Tris pH 7.4100 mmol/L HEPES pH 7.4100 mmol/L MOPS pH 7.4100 mmol/L 醋酸钠pH 4注意：1.为了最佳的操作过程，按照上述纯化步骤进行预洗以除去任何削弱结合镍离子的物质。
anti-flag亲和纯化凝胶纯化纳微
亲和纯化是一种常见的生物化学技术，用于从复杂混合物中纯化特定的生物大分子，比如蛋白质、DNA或RNA等。
而 anti-flag 亲和纯化就是利用与Flag标签结合的抗体进行纯化的一种方法。
本文将为您介绍 anti-flag 亲和纯化凝胶纯化纳微的相关知识和技术原理。
一、anti-flag 亲和纯化的原理1.1 Flag 技术的概念Flag 标签是一种广泛应用于蛋白质生物化学和细胞生物学研究中的蛋白质标记技术。
它是一种八个氨基酸残基的片段，可以与抗Flag抗体特异性结合，在一系列生物学实验中得到了广泛应用。
1.2 anti-flag 亲和纯化的原理anti-flag 亲和纯化是利用特异性结合于Flag标签的抗体，将Flag标记的蛋白从混合物中纯化出来的一种技术方法。
该方法利用了抗体与抗原结合的高亲和性，使得目标蛋白可以在保持其生物活性的情况下被特异性地纯化。
二、anti-flag 亲和纯化凝胶纯化纳微的步骤2.1 样品制备首先需要准备包含Flag标记蛋白的混合物样品，该样品可以是原核或真核细胞的裂解液，也可以是细胞培养上清中的蛋白混合物。
2.2 抗体固定将抗Flag抗体固定在凝胶上，使其形成亲和纯化凝胶。
2.3 样品加载将样品加载到亲和纯化凝胶柱中，使其中的Flag标记蛋白与固定的抗体结合。
2.4 洗脱步骤通过洗脱步骤，去除非特异性结合蛋白和杂质。
2.5 Elution 步骤通过适当的条件，比如改变 pH 或者添加特定的化合物，使得所需的Flag标记蛋白脱离抗体从而得到纯净的目标蛋白。
三、anti-flag 亲和纯化凝胶纯化纳微的应用3.1 蛋白纯化anti-flag 亲和纯化凝胶纯化纳微常用于蛋白质的纯化，尤其是对于标记有Flag tag 的蛋白质。
3.2 细胞信号通路研究在细胞信号通路研究中，常常需要对Flag标记的蛋白进行纯化，以进行其生物学功能的研究。
3.3 转基因动物研究在转基因技术中，为了对转基因蛋白进行研究，需要将其从复杂细胞组分中纯化出来，而anti-flag 亲和纯化凝胶纯化纳微则提供了一种高效、特异性的方法。
心型脂肪酸结合蛋白(h-FABP)ELISA试剂盒说明书
心型脂肪酸结合蛋白(h-FABP)酶联免疫分析试剂盒预期应用ELISA法定量测定人血清、血浆或其它相关生物液体中心型脂肪酸结合蛋白(h-FABP)含量。
实验原理用纯化的抗体包被微孔板，制成固相载体，往包被抗h-FABP抗体的微孔中依次加入标本或标准品、生物素化的抗h-FABP抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。
TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。
颜色的深浅和样品中的h-FABP呈正相关。
用酶标仪在450nm波长下测定吸光度（OD值），计算样品浓度。
试剂盒组成及试剂配制1. 酶联板：一块（96孔）2. 标准品（冻干品）：2瓶，每瓶临用前以样品稀释液稀释至1ml，盖好后静置10分钟以上，然后反复颠倒/搓动以助溶解，其浓度为150 ng/ml，请先稀释至30 ng/ml，再做系列倍比稀释(注：不要直接在板中进行倍比稀释)后，分别稀释成30 ng/ml，15 ng/ml，7.5 ng/ml，3.75 ng/ml，1.88 ng/ml，0.94 ng/ml，0.47 ng/ml，样品稀释液直接作为标准浓度0 ng/ml，临用前15分钟内配制。
如配制15 ng/ml标准品：取0.5ml (不要少于0.5ml )30 ng/ml的上述标准品加入含有0.5ml样品稀释液的Eppendorf管中，混匀即可，其余浓度以此类推。
3. 样品稀释液：1×20ml。
4. 检测稀释液A：1×10ml。
5. 检测稀释液B：1×10ml。
6. 检测溶液A：1×120μl（1:100）临用前以检测稀释液A 1:100稀释，稀释前根据预先计算好的每次实验所需的总量配制（100μl/孔），实际配制时应多配制0.1-0.2ml。
如10μl 检测溶液A加990μl检测稀释液A的比例配制，轻轻混匀，在使用前一小时内配制。
7. 检测溶液B：1×120μl/瓶（1:100）临用前以检测稀释液B 1:100稀释。
Flag标签亲和层析介质
Flag标签亲和层析介质Anti-flag Affinity Beads货号规格BDTL0045-11mlBDTL0045-55ml1.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-flag 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-flag 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-flag Affinity Beads产品性能项目性能基质4%琼脂糖微球配体Anti-flag鼠单克隆抗体载量>1mg flag标签蛋白/ml介质粒径(μm)45-165最大流速0.1MPa,1bar储存缓冲液0.02%叠氮化钠，1XPBS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0.45μm滤膜过滤。
平衡和洗杂液:50mM Tris,0.15M NaCl,pH7.4洗脱液:0.1M glycine HCl,pH3.5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，pH8.0再生液1：0.1M Tris-HCl,0.5M NaCl,pH8.0-1-再生液2：0.1M NaAc,0.5M NaCl,pH4.02.2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-flag 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。
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。
FLAG HA双重亲和性诱导净化套件说明书
FLAG®HA Tandem Affinity Purification KitCatalog Number TP0010Storage Temperature 2-8 °CTECHNICAL BULLETINProduct DescriptionThe FLAG HA Tandem Affinity Purification Kit is designed for the isolation of high purity FLAG-HA dual-tagged fusion proteins from complex matrices, such as cell lysates and tissue homogenates. The kit is particularly suitable for isolation of protein complexes using TAP (Tandem Affinity Purification) technology. TAP technology is a recent development in protein purification. This technology incorporates tandem-linked affinity tags into genes of interest so that fusion proteins can be isolated through two consecutive affinity purification steps. The technology is particularly useful for the isolation and identification of protein complexes by adding a tandem affinity tag to a known “bait” protein to pull down endogenous proteins that interact with the targeted protein of interest. Protein complexes isolated using TAP technology have a much higher purity compared to single purification and can be used for mass spectrometry (MS) analysis .1,2Although existing TAP systems using combinations of GST, calmodulin, nickel binding peptides, streptavidin, and Protein A have been used in various experimental systems, they suffer from several limitations: interference with complex assembly or protein function due to relatively large TAP tags (20kDa and larger), high rate of cross reactivities with non-targeted endogenous proteins, and/or elution requirement of TEV protease.The FLAG HA tandem epitope tagging system eliminates or minimizes these concerns. Both FLAG (DYKDDDDK)and HA (YPYDVPDYA)are small, non-eukaryotic derived tags. These features minimize interference with protein functions and provide superior specificity. The hydrophilic character of the FLAG HA peptides increases the likelihood that the dual epitope will be located on the surface of the fusion protein where it is accessible for antibody-antigen interaction. Antibodies against FLAG and HA tags and affinity resins derived from them have demonstrated a high specificity and affinity in pull-down assays compared to other existing epitope-tagging systems.As shown below, the protein purification procedure using the FLAG HA Tandem Affinity Purification Kit is simple and fast. The kit can be used for isolation of single tandem-tagged fusion proteins or for isolation of protein complexes by co-immunoprecipitation using tandem-tagged “bait” proteins.Outline of FLAG HA Fusion Protein Purification Precautions and DisclaimerFLAG HA Tandem Affinity Purification kit is for R&D use only, not for drug, household or other uses. Consult the MSDS for information regarding hazards and safe handling practices.Storage/StabilityThe kit is shipped on wet ice. Upon receipt, store the spin columns and collection tubes at room temperature. Store the remaining reagents at 2-8 °C.12(Different elution strategies depending onPrepare Cell Lysate(Presence of a FLAG-HA fusion protein required)Bind to ANTI-FLAG resin(Sample volume can be scaled up to 10 ml or larger)Transfer and Wash Resin(Transfer resin to a Spin column and wash)st Elution of FLAG-HA-tagged Protein(Competitive elution with 3x FLAG peptide)Bind to Anti-HA resin(Incubate in a Spin column)Wash Resinnd Elution of Protein/protein Complexdownstream applications)2Table 1Additional Reagents and Equipment Not Supplied • FLAG HA TAP Tag Generation Kit, Product CodeTP0020• Immunoprecipitation compatible extraction buffer • Protease inhibitor cocktail (see Table 2)• Conical vials and microcentrifuge tubes • Pipettes and tips• Refrigerated microcentrifuge • Shaker/agitator• Tris Buffered Saline TBS (0.138 M NaCl, 0.003 MKCl, 0.05 M Tris; pH 8.0)Product Code T6664• Water, Molecular Biology Reagent, Product CodeW4502o HA peptide, Product Code I2149o 2X Laemmli Sample Buffer (LSB), Product CodeS3401o Ammonium bicarbonate, Product Code A6141o C18 micro spin column (Vivapure ®fromVivascience)o Chemiluminescent Peroxidase Substrate-1,Product Code CPS1120o Monoclonal ANTI-FLAG ®M2-Peroxidase (HRP),Product Code A8592o Monoclonal Anti-HA-Peroxidase (HRP), ProductCode H6533o ProteoSilver ™Plus Silver Stain Kit, Product CodePROTSIL2˜required šoptionalGeneral NotesPlease read the entire protocol before proceeding with the procedure.Extract PreparationThe appropriate amount of starting material depends on the downstream protein analysis technique intended. The abundance of the tagged protein and theinteracting endogenous complexes will also determine the necessary amount of starting material. As a frame of reference, 15-50 g of transgenic Arabidopsisseedlings (fresh weight) and 1 X107transfected COS-7 cells have been used successfully to isolate tandemly tagged protein for visualization and identification. The concentration of protein in the extracts should be 1-100mg/ml. For protein identification by massspectrometry (MS) and Edman sequencing, the protein concentration is measured in picomoles (pmols) and not micrograms (µg). The following formula can be used for picomole/microgram conversion:1000/MW of the protein in kDa = pmol/µgExtracts should be prepared in an immunoprecipitation-compatible buffer with added protease inhibitors (see table below). Removal of cellular debris by multiple filtration steps or by centrifugation is necessary. If protein-protein interactions mediated byphosphorylation are being investigated, suitablephosphatase inhibitors (such as P5726 and/or P2850) should be included. When performing experiments, samples should remain at 4 °C when possible.Table 23EZview ANTI-FLAG M2 and anti-HA affinity resinsThe ANTI-FLAG and anti-HA affinity resins have been created by covalently linking monoclonal antibodies to resin and are supplied as a 50% slurry suspension in phosphate buffered saline with an anti-microbial agent. The initial immunoprecipitation volume is relatively large and dependent on the experimental design. EZview ANTI-FLAG resin is provided to enhance visibility during incubation and transfer manipulations. The red color of EZview resin does not affect binding capacity. Using epitope tagged proteins for quantification, ANTI-FLAG and anti-HA resins were found to have binding capacities greater than or equal to 0.6mg/ml and 0.4mg/ml, respectively.There are many variations in immunoprecipitation procedures. The initial incubation volume affects the amount of ANTI-FLAG resin used. The user may need to adjust the amount of resin used to satisfy the specific experimental requirements. In general, 100µl of ANTI-FLAG resin slurry is appropriate for 10ml of extract. Approximately 300 to 500 µl of ANTI-FLAG resin is recommended for 30 ml of extract. Larger extract volumes (>30 ml) should be divided for processing. Carefully mix resins for homogeneous suspension before aliquots are taken. Resins should be washed 3X with the supplied RIPA buffer. The volume of eachwash should be at least 4X the packed-resin volume. In case of numerous immunoprecipitation samples, wash the resin needed for all the samples together. After washing, divide the resin according to the number of samples. Use a pipette tip with a wide bore to dispense slurry. Centrifugation should be done at 3,000 X ing SigmaPrep spin columns with breakable tips The use of spin columns ensures minimal loss of the affinity resins during washing. Break off the end tip of the spin column and plug column with tip. See Figure 1. The column has a capacity of 750 µl. Keep the tipinserted in the bottom of the column and the red screw cap secure on the column during the incubation steps. Remove the tip from the column during centrifugation and save for later steps. The column should be placed in a 2 ml collection tube for the washing step, or a microcentrifuge tube for the elution step. The columnand red screw cap should be labeled.ProcedureStep 1. Binding to ANTI-FLAG M2 resinA.Prepare RIPA + protease inhibitor (PI) cocktail (see Table 2 for suitable inhibitors). Each sample needsapproximately 4 ml of RIPA + PI (for Step 2.A, 2.B, 4.B, and 5.B).B.Aliquot an appropriate amount of ANTI-FLAG M2 resin into a clean tube or vial. Wash three times with RIPA buffer. For each of the washes, gently agitate the resin in the buffer then centrifuge at 3,000 X g for 30 seconds. Decant as much of the remaining final wash volume as possible without losing any resin. (See General notes on affinity resins for amount of ANTI-FLAG resin/sample and resin-washing directions.)C.At this point, a FLAG HA fusion protein must be present in the extract. It is worthwhile to confirm the presence of a FLAG HA protein with an appropriate immunodetection technique. If the FLAG HA protein is not endogenously expressed in the tissue or species being investigated, it is necessary to add purified FLAG HA protein to experimental samples to serve as a “bait” protein.Add washed ANTI-FLAG ®M2 resin (from Step B) to the sample extract (See Extract Preparation under General Notes). Conical vials may be used as incubation containers.D.Incubate two hours to overnight at 4 °C with gentle rocking or agitation. The resin must remain suspended during the incubation.4Step 2. Removing unbound protein.A.Spin the conical vial (from Step 1.D.) at 3,000 X g at4 °C for two minutes. The supernatant containsunbound protein. Remove as much of the supernatantas possible with a pipette without disturbing the resin.Discard the supernatant. Again, using a pipette, carefully transfer the remaining supernatant and resininto a spin column that has been placed into acollection tube. For adequate washing, the spincolumn’s packed-resin volume should not exceed 150 µl. Use multiple spin columns if needed. Add 500µl RIPA + PI cocktail to each spin column being used. Spin sample in microcentrifuge at 3,000 X g at 4 °C for30 seconds. (See page 3 for notes on using SigmaPrepspin columns).B.Reinsert tip into column(s) (See Fig. 1). Add 500 µlof RIPA + PI cocktail. Agitate samples for several minutes at 4 °C.C.Remove tip from column(s). Spin column(s) in the same 2 ml collection tube at 3,000 X g at 4 °C for30seconds. Discard the column washings whichcontains unbound proteinD.Repeat wash steps (B-C) two more times.Step 3. First elution of the protein complex (using3XFLAG peptide)A.Reinsert tip into column(s). Place column(s) intoclean microcentrifuge tube(s).B.Preparing the 3XFLAG peptide:Prepare a 5mg/ml 3XFLAG peptide stock solution by adding TBS, Product Code T6664, to the product vial and dissolve powder by vortexing. Aliquot and store at −20 °C. Repeated freezing and thawing is not recommended.For elution, add 3 µl of 5µg/µl 3XFLAG peptide stock solution to 100 µl of TBS (150 ng/µl final concentration).C.Elute the tandem tagged FLAG HA protein with2.5times the packed-resin volume of 150 ng/µl3XFLAG peptide (see Table 3). Incubate resin/elutionvolume for at least 10 minutes. The resin must remainsuspended during the incubation.D.Remove the tip and spin the column in a clean microcentrifuge tube at 3,000 X g for 1minute. Keep the eluate, which contains the eluted protein.E.Perform a second elution (Steps C-D) and pool the eluates.Table 3Step 4. Binding to anti-HA resinA.Determine the number of new spin columns needed from Table 3. Use 40 µl of washed anti-HA resin slurry for each column. See General notes on affinity resins for resin-washing directions.Final elutions can be concentrated by not splitting larger volumes (≥750 µl) into multiple spin columns and not increasing resin amount. During the anti-HA resin incubation (Step 4.B.), use a microcentrifuge tube instead of a spin column. After incubation with anti-HA resin, the sample needs to be sequentially added to the spin column (Step 5.A.).B.Break the tip(s) off new column(s), invert, and plug column(s) (See Fig. 1). Add xµl RIPA +PI, 20 µl packed-resin volume of washed anti-HA resin, and the 3XFLAG peptide elution (from Step 3). The maximum volume of the column is 700 µl. Incubate the column from 30minutes to 2 hours at 4°C with gentle rocking or agitation. The resin must remain suspended during the incubation.Step 5. Removing unbound proteinA.Remove tip and place the spin column(s) into a new 2 ml collection tube. Spin at 3,000 X g for 30 seconds without tip. Discard column washings..B.Reinsert column tip. Add 500µl of RIPA + PI cocktail. Agitate samples for several minutes. If the eluted sample will be assayed directly by mass spectrometry without running PAGE (see information under Step 6.A.3.) wash in TBS instead of RIPA. RIPA contains detergents that interfere with MS results; however, residual amounts of RIPA enhance urea elution.C.Remove the tip and spin column(s) in the same 2 ml collection tube at 3,000 X g for 30 seconds. Discard column washings, which contains unbound protein.D.Repeat wash steps (B-C) two more times.5Step 6. Final elution of the protein complexThree elution methods are recommended according to downstream applications and preference.A.Elution with ureaFor MS analysis of the protein, elute the sample with8M urea. Urea will preferentially elute the prey proteins that interact with the “bait” (dual tagged) protein. See Figure 2. This maximizes the detection sensitivity of unknown proteins by reducing contamination by the “bait” protein. The protein is denatured. Urea elution is applicable to PAGE gel and direct MS submission1.Add 16 ml of water (W 4502) to the bottlecontaining urea and vortex. The resulting solutionwill be 25 ml of 8 M urea.2.Insert tip into column outlet. Put the column withresin sample into a new microcentrifuge tube.Elute 20 µl of packed-resin from Step 5.D. with 50 µl of 8 M urea at room temperature. Incubateresin/elution volume for a minimum of 10 minutes.The resin must remain suspended during theincubation. Remove tip from column. Spin at 3,000 X g for 1minute. Sample can now be prepared to load on a PAGE gel.3.For direct MS submission, samples needed to bewashed with TBS in Step 5.B. Protein needs to be at a picomole level (See General Notes on Extract Preparation on page 2). Dilute the eluate (as inStep 6.A.2.) with 100mM ammonium bicarbonateuntil the final urea concentration is ≤1M. Sample is then ready to be digested with trypsin. For MSanalysis, the sample can be concentrated using aC18 micro spin column (such as Vivapure fromVivascience). Figure2: Urea preferentially elutes the prey complex from the bait protein attached to the resin. p53 and large T-antigen interact in COS-7 cells (a mammalian cell line). FLAG HA was incorporated into p53 using overlapping PCR (see FLAG HA Tandem TAP Tag Generation Kit TP0020), placed into an expression vector driven by the CMV2 promoter, and transiently expressed. Transfected and non-transfected samples were processed using ANTI-FLAG resin followed by anti-HA resin. Protein was eluted with 3XFLAG peptide (A-once, B-twice) from the ANTI-FLAG resin then urea (C) followed by LSB (D) from the anti-HA resin. Resins were washed with RIPA through out the procedure. The prey protein is predominantly eluted in urea, while the bait protein is not.B.Elution with HA peptide (I 2149).This elution strategy is the mildest elution, suitable for recovery of single dual-tagged proteins or protein complexes. HA peptide competitively elutes the tandem tagged protein from the HA resin with minimum interference to protein function.1.Prepare the HA peptide (I2149, not supplied)according to the product instructions. The finalworking dilution should be 1 µg/µl.2.Insert tip into column outlet. Put the column withresin sample into a new microcentrifuge tube.Elute 20 µl of packed-resin from Step 5.D. with 50µl of 1 µg/µl HA peptide. Incubate for at least 10minutes. The resin must remain suspended during the incubation. Remove tip from column. Spin at3,000 X g for 1 minute. Save eluate in amicrocentrifuge tube. Repeat elution. Combine theeluates.PreyDetecting12BaitFLAG HA p53T-antigenBait presence---++++st IP (FLAG)nd IP (HA)A B C DElutionsFigure 26C.Elution with 2X Laemmli Sample Buffer (LSB) Product Code S3401This is a harsh elution condition that elutes everything, including the antibody from the resin. It is useful for analysis of tagged proteins and protein complexes by PAGE gel and Western blotting.1.Insert tip into column outlet. Put the column withresin sample into a new microcentrifuge tube.Elute 20 µl of packed resin (from Step 5.D) with50µl 2X LSB. Incubate and mix for 5 to 10 minutes.The resin must remain suspended during theincubation. Remove plug from column. Spin at3,000 X g for 1minute. The eluate contains thesample. Heat sample at 95 °C for 5 minutes.2.Run on a PAGE gel. Heavy and light chains of theantibody (from the HA resin) are present and mayinterfere with visualizing the proteins of interest. References1.Honey, S., et al., A novel affinity purification tag andits use in identification of proteins associated with a cyclin-CDK complex. Nucleic Acids Res., 29, e24(2001).2.Rigaut, G., et al., A generic protein purificationmethod for protein complex characterization andproteome exploration. Nat. Biotechnol. 17, 1030 -1032 (1999).3.Rose, J. K. C., et al., Tackling the plant proteome:practical approaches, hurdles and experimentaltools. Plant J.,39, 715-733 (2004).4.Shevchenko, A., et al. Deciphering proteincomplexes and protein interaction networks bytandem affinity purification and Mass Spectrometry.Mol. Cell. Proteomics, 1, 204-212 (2002).5.Hall, D. B., and Struhl, K., The VP16 ActivationDomain Interacts with Multiple TranscriptionalComponents as Determined by Protein-ProteinCross-linking in vivo. J. Biol. Chem., 277, 46043-46050 (2002).7 Troubleshooting GuideIGEPAL is a registered trademark of Rhodia OperationsVivapure is a Registered Trademark of Vivascience AGFLAG, ANTI-FLAG, and 3XFLAG are Registered Trademarks of Sigma-Aldrich Biotechnology LLCEZview, ProteoSilver, FLAG-BAP, and SigmaPrep are trademarks of Sigma-Aldrich Biotechnology LLCAH,RS,PHC 08/10-1Sigma brand products are sold through Sigma-Aldrich, Inc.Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side ofthe invoice or packing slip.。
GST蛋白纯化试剂盒说明书及操作指南
GST蛋白纯化试剂盒说明书及操作指南GST蛋白纯化试剂盒是一种常用的实验工具，用于从细胞裂解液中高效纯化谷胱甘肽S转移酶（GST）标签融合蛋白。
下面将详细介绍试剂盒的组成、使用步骤以及注意事项，以帮助用户顺利进行GST蛋白纯化实验。
一、试剂盒组成蛋白纯化试剂盒由以下几个主要组分组成：1、离心管：提供用于样品处理和离心过程的离心管。
2、细胞裂解缓冲液：含有适量的洗脱缓冲液和辅助物质，在细胞裂解过程中保护目标蛋白的完整性。
3、离心管悬浮粉末：用于牢固固定GST亲和树脂。
4、清洗缓冲液：用于去除非特异性结合物质，保证目标蛋白的纯度。
5、洗脱缓冲液：用于洗脱目标蛋白，使其从GST亲和树脂上解离。
二、操作步骤以下是使用GST蛋白纯化试剂盒进行GST蛋白纯化的基本步骤：1、细胞裂解：将经过表达的GST标签融合蛋白的细胞用细胞裂解缓冲液裂解，并加入适量的离心管悬浮粉末。
轻轻摇晃或旋转离心管，使粉末均匀分布。
2、离心：将裂解液转移至离心管中，并以适当的速度离心使GST亲和树脂沉淀到离心管底部。
3、除去上清：谨慎倒出上清液，避免破坏沉积的GST亲和树脂。
4、清洗：加入适量的清洗缓冲液，轻轻摇晃离心管，使清洗缓冲液与GST 亲和树脂充分接触，去除非特异性结合物质。
重复此步骤两次以确保清洗。
5、洗脱：加入适量的洗脱缓冲液，轻轻摇晃或旋转离心管，使目标蛋白从GST亲和树脂上洗脱。
6、收集洗脱液：将洗脱液收集于新的离心管中，这样就得到了纯化的GST 标签融合蛋白。
三、注意事项在使用蛋白纯化试剂盒过程中，请注意以下几点：1、严格按照说明书中的步骤进行操作，避免操作失误导致结果不准确。
2、所有悬浮粉末和缓冲液应按照说明书中的要求储存，并避免暴露在高温、阳光直射或湿度过高的环境中。
3、使用前请检查试剂盒是否有损坏或过期，如有问题请勿使用。
4、操作过程中请佩戴适当的实验防护设备，避免对自己和他人造成伤害。
准确使用GST蛋白纯化试剂盒可以高效地纯化目标蛋白，为科研工作者提供了一个有效的实验工具。
FLAG标签融合蛋白纯化试剂盒使用说明书
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。
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 .。
融合亲和蛋白试剂安全操作及保养规程
融合亲和蛋白试剂安全操作及保养规程前言融合亲和蛋白试剂是一种常见的蛋白质纯化试剂，能够选择性地结合目标蛋白质从而进行纯化。
然而，在使用融合亲和蛋白试剂的过程中，需要注意其安全操作与保养，以确保实验的准确性和人员的安全。
本文将就融合亲和蛋白试剂的安全操作及保养规程进行详细地阐述。
安全操作1、个人防护在操作融合亲和蛋白试剂时，需要穿戴适当的实验室防护服，并戴上手套、口罩、护目镜等个人防护用具，以防止试剂溅到皮肤或吸入对呼吸道造成的损害等。
入室前要洗手，并保持好个人卫生。
2、试剂储存融合亲和蛋白试剂应储存在阴凉干燥处，避免阳光直射。
不要将试剂放在高温环境或者阳光下，以免造成试剂变质或损坏。
3、实验前准备在进行融合亲和蛋白实验之前，需要先完成实验室的环境准备，保持实验室的整洁，并放置好所需的试剂、器材等。
实验前应核对试剂品名、编号等信息，避免因为规格不符或编号错误导致的试验失败或压缩产物质量。
4、实验中操作在取用融合亲和蛋白试剂时应遵循实验室安全规范，避免发生噴溅或中毒等意外。
试剂使用过程中，应进行标记，方便管理和识别，同时要避免混淆或误用。
操作过程中，需要保持实验台干净整洁，确保实验品质。
5、废弃物处理实验结束后，废弃物需要按照实验室规定进行处理。
融合亲和蛋白试剂通常以废液的形式排放，因此废液的安全处理至关重要。
废液应按照实验室规定的方式进行分类、置于特定容器内收集，倾倒时要注意不要与其他废液混合。
保养规程1、装箱保藏打开融合亲和蛋白试剂包装时，需要注意不要弄坏试剂包装袋。
实验未用完的融合亲和蛋白试剂，需要放回包装袋并尽快上封口，防潮密封。
保存好的试剂在温度适宜的情况下可以得到长期保存。
融合亲和蛋白试剂袋在不使用时要尽量保持密封，避免湿气和细菌感染。
2、常规检测时常对储存的融合亲和蛋白试剂进行常规检测，比如浓度、pH值和酶活性等指标检测，以确保试剂还可以继续使用。
如发现某些参数不稳定，须重新参考说明书须配制新的试剂。
flag标签蛋白纯化原理
flag标签蛋白纯化原理
flag标签是一种常用的蛋白质标签，它由8个重复的氨基酸序列DYKDDDDK组成，可以在目的蛋白质的N或C端加入。
这种标签可以用于蛋白质的定位、鉴定和纯化等方面，具有简单、高效、方便的特点。
flag标签蛋白纯化的原理是利用flag标签与抗flag抗体之间的特异性结合，将包含flag标签的蛋白质特异地捕获到固定在固相上的抗flag抗体上，实现蛋白质的纯化。
在纯化过程中，可分别采用亲和层析、免疫沉淀等方法进行。
flag标签蛋白的纯化过程主要包括以下几个步骤：
1.制备含有flag标签蛋白的细胞提取物或培养上清液。
2.将细胞提取物或培养上清液加入固相上的抗flag抗体。
抗体可固定在各种载体上，如琼脂糖、磁珠等。
3.通过洗涤和洗脱等步骤，将非特异性结合的蛋白质去除，留下特异性结合的flag标签蛋白。
4.进行浓缩、纯化等后续处理，得到高纯度的flag标签蛋白。
flag标签蛋白纯化的优点在于操作简便、可重复性好、产量高、效果稳定等，是一种常用的蛋白质纯化方法。
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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 mM
NaCl, 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。
冰上
放置30min之后，12000rpm，4℃离心10min。
取上清，冷冻保存。
4.2装柱及孵育
4.2.110ml（10倍柱体积）PBS清洗Anti-Flag亲和纯化柱。
4.2.2加入含有目标蛋白的真核细胞裂解液，室温孵育或者4℃孵育过夜。
4.2.3根据蛋白质性质选择洗脱方法，具体可参见第5部分问题和建议
5.1。
4.33xFlag peptide洗脱液竞争洗脱
4.3.1用1xPBS配制3×Flag peptide洗脱液，终浓度为100μg/ml。
4.3.2加入10ml 3×Flag peptide洗脱液（10倍柱体积）进行洗脱，每次收集
1ml。
4.3.3用紫外检测仪测定收集液, 合并收集液。
4.3.4SDS-PAGE鉴定蛋白质纯度及浓度，并按照需求处理和保存蛋白质。
4.4酸性洗脱液酸性洗脱
4.4.15ml PBS（5倍柱体积）洗柱三次。
4.4.2预冷的5ml pH
5.0酸性预洗液（5倍柱体积）洗涤纯化柱，除去非特异性
结合蛋白。
4.4.3预冷的10ml pH 3.0的酸性洗脱液（10倍柱体积）进行洗脱，每次收集
1ml, 收集管中预先放入中和液50μl。
注意：酸性环境放置太久会缩短亲和
纯化柱的使用寿命，应尽量缩短亲和纯化柱与酸性洗脱液的接触时间。
4.4.4用紫外检测仪测定收集峰, 合并收集峰。
4.4.5SDS-PAGE鉴定蛋白质纯度及浓度，并按照需求处理和保存蛋白质。
4.5纯化柱的清洗与再生（洗脱后须立即进行纯化柱的清洗与再生）
4.5.110倍柱体积PBS清洗纯化柱。
4.5.2酸性洗脱液（pH3.0）洗涤纯化柱，每次三倍柱体积。
4.5.3中和液（pH8.0）洗涤柱子三次，每次三倍柱体积。
4.5.4检测流穿液pH，如为中性，则进行下一步；如仍为酸性，则重复4.5.2和
4.5.3。
4.5.5用1xPBS（含50%甘油，0.2‰叠氮钠）清洗，再加入适量该保存溶液至
填料中，保存在20℃。
4.6保存
纯化产物加入50%甘油保存在-80度，用于后续功能性实验。
5.问题与建议
5.1如何选择竞争性洗脱和酸性洗脱？
3×Flag peptide洗脱液与蛋白质上的Flag标签竞争亲和纯化柱上的抗体，使蛋白质与抗体的结合被取代而脱离纯化柱，从而被洗脱下来。
这种洗脱的特点有：
5.1.1洗脱条件温和，一般不会造成蛋白质变性；有利于亲和纯化柱的保存和反复
使用。
5.1.2由于亲和吸附达到平衡比较慢，所以往往需要较常的时间和较大的洗脱体积。
5.1.3特异性强，可以进一步消除非特异性吸附的影响，从而得到较高的分辨率。
基本上不会有anti-Flag的抗体被洗脱下来。
酸性洗脱是通过改变环境pH，破坏抗原抗体的结合从而使蛋白质脱离纯化柱被洗脱下来，这种洗脱方式的特点有：
5.1.4成本低廉。
5.1.5纯化速度较快。
5.1.6酸性pH会造成部分蛋白质变性，引起目的蛋白质沉淀，降解或失活。
5.1.7有时会有少部分Anti-Flag抗体被洗脱，造成非特异性信号。
5.1.8反复使用酸性洗脱，会造成抗体脱落，也会破坏纯化柱的理化性质，减少纯
化柱的反复使用次数。
5.1.9有文献显示，与传统的Glycine-HCl洗脱液相比，本试剂盒提供的pH3.0
的Arginine-HCl做为洗脱液，可以减少蛋白质变性，延长抗体亲和纯化柱的
使用寿命。
客户可以根据实际情况自行选用配制。
5.2常见的蛋白质的后续处理方法有哪些？
5.2.1透析与超滤
透析法是利用半膜将分子大小不同的蛋白质分开。
超滤法是利用高压力或离
心，强使水和其他小的溶质分子通过半透膜而蛋白留在超滤管内。
两者都可
以达到换液的目的。
使用时需注意选择正确分子量的透析袋和超滤管。
5.2.2过滤除菌
可采用微孔滤膜滤器操作，让蛋白质溶液通过0.22um滤膜，即可达到除菌
的目的。
5.2.3蛋白质定量鉴定浓度。
5.2.4SDS-PAGE鉴定纯度。
5.3如何选择细胞/组织裂解液？
本试剂盒提供的细胞裂解液成分如下：
Nacl 150mM
Tris-HCL(PH8.0) 50 mM
TRITON X-100 1%
EDTA 1mM
使用前可加入1%PMSF和0.5%原钒酸钠, 或其他蛋白酶抑制剂，避免蛋白质降
解。
客户可根据目的蛋白的性质和后继用途，自行配制细胞裂解液及选择是否加入蛋白酶抑制剂。
6.使用中的常见问题
来源出处：武汉戴安生物技术有限公司。