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 Protein A+G Agarose (Fast Flow, 进口分装)

  产品编号P2012
  产品包装: 2ml
  产品价格: 501.00元
产品编号 产品名称 产品包装 产品价格
P2012 Protein A+G Agarose (Fast Flow, 进口分装) 2ml 501.00元

      本Protein A+G Agarose (Fast Flow)为进口分装,主要用于免疫沉淀(Immunoprecipitation,IP)或免疫共沉淀(Co-IP),也可以用于抗体的纯化。
      Protein A+G Agarose适合于免疫沉淀所有Protein A Agarose和Protein G Agarose单独可以免疫沉淀的抗体,包括mouse IgG1, IgG2a, IgG2b, IgG3, IgA, rat IgG1, IgG2a, IgG2b, IgG2c, rabbit IgG, rabbit and goat polyclonal Abs,以及human IgG1, IgG2, IgG3和IgG4
      Protein A和Protein G都共价交联到4% agarose beads (Fast Flow)上,2ml Protein A+G Agarose中共含有约2mg重组的Protein A+G。2 ml Protein A+G Agarose共可以结合约15mg human IgG。推荐的线性流速(Linear flow rate)为 50-
300cm/h。
      Protein A+G Agarose配制在TBS溶液中,2ml中共含有0.5ml Agarose beads。 
      本Protein A+G Agarose如果用于常规的免疫沉淀,可以免疫沉淀100次。
包装清单:

产品编号 产品名称 包装
P2012 Protein A+G Agarose (Fast Flow, 进口分装) 1ml×2
说明书 1份

保存条件:
      4℃保存,一年有效。
注意事项:
      请勿冷冻保存本产品。
      Protein A+G Agarose使用前一定要充分重悬,即充分颠倒若干次使混合均匀。
      从蛋白样品收集开始,所有步骤中蛋白样品都必须在4℃或冰上操作。
      本产品仅限于专业人员的科学研究用,不得用于临床诊断或治疗,不得用于食品或药品,不得存放于普通住宅内。
      为了您的安全和健康,请穿实验服并戴一次性手套操作。 

使用说明:
1. 免疫沉淀(Immunoprecipitation,IP):
a. 蛋白样品的准备:
(a) 对于10厘米细胞培养皿中的贴壁细胞,吸除细胞培养液,PBS洗涤一次,然后加入500微升至2毫升细胞裂解液裂解细胞。可以使用碧云天生产的Western及IP细胞裂解液(P0013)或各种RIPA裂解液(P0013B、P0013C、P0013D或P0013E)等进行细胞的裂解。
(b) 对于组织样品参考贴壁细胞使用裂解液的比例进行裂解。
(c) 对于悬浮细胞,离心收集细胞后,PBS洗涤一次,然后参考贴壁细胞的裂解方法进行裂解。
注: 详细的裂解方法参考不同裂解液的详细使用方法。对于不同的培养器材,参考10厘米培养皿的裂解液的用量进行裂解。如果裂解获得的蛋白样品浓度过高,可以用裂解液或PBS适当稀释,如果蛋白样品浓度过低,在以后的裂解过程中宜适当减少裂解液的用量。
b. 去除非特异性结合(可选做)
(a) 取200微升至1毫升蛋白样品,蛋白量约为200微克至1毫克,加入约1微克和免疫沉淀时使用的IgG种属相同的普通IgG和20微升充分重悬的Protein A+G Agarose,4℃缓慢摇动30分钟至2小时。
(b) 2500rpm(约1000g)离心5分钟,取上清用于后续的免疫沉淀。
注:所谓种属相同的IgG是指,例如后续免疫沉淀时用的是小鼠IgG,则在本步骤中可以加入normal mouse IgG,如无normal IgG可以加入其它不影响后续检测的其它mouse IgG类型的抗体。通过和normal IgG和Protein A+G Agarose的孵育,可以充分降低非特异性的结合,降低背景。
c. 免疫沉淀:
(a) 加入0.2-2微克用于免疫沉淀的一抗,4℃缓慢摇动过夜。
(b) 再加入20微升充分重悬的Protein A+G Agarose,4℃缓慢摇动1-3个小时。(为方便后续的洗涤操作可以把加入充分重悬的Protein A+G Agarose的量调整为40微升。)
(c) 2500rpm(约1000g)离心5分钟,或瞬时高速离心,小心吸除上清,注意宁可留下少量上清也不能吸掉Protein A+G Agarose。
(d) 用准备蛋白样品时的裂解液或PBS洗涤沉淀5次,裂解液或PBS的用量每次为0.5-1毫升。洗涤时离心条件和吸除上清的要求同上面的步骤c(c)。
(e) 完成最后一次洗涤后,去除上清,加入20-40微升1XSDS-PAGE电泳上样缓冲液Vortex重悬沉淀,瞬时高速离心把样品离心至管底。
(f) 100℃或沸水浴处理3-5分钟,取部分或全部样品用于SDS-PAGE电泳,暂时不用的样品可以-20℃保存。
2. 免疫共沉淀:
参考免疫沉淀的方法进行,但免疫共沉淀(co-IP)通常必须使用未经冻存的新鲜蛋白样品。普通的免疫沉淀虽然可以使用冻存的蛋白样品,但也宜用新鲜的蛋白样品为佳。

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