碧云天生物技术-Protein A Agarose (Fast Flow, 进口分装)(P2006)

试剂> 多肽与蛋白> 蛋白检测> 免疫沉淀

Protein A Agarose (Fast Flow, 进口分装)

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产品编号: P2006

产品包装:2ml

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价格: ¥ 497.00

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产品编号	产品名称	产品包装	产品价格
P2006	Protein A Agarose (Fast Flow, 进口分装)	2ml	497.00元

本Protein A 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 Agarose适合于免疫沉淀mouse IgG_2a, IgG_2b, IgA, rabbit IgG, 以及human IgG₁, IgG₂和IgG₄。
Protein A共价交联到6% agarose beads (Fast Flow)上，2ml Protein A Agarose中共含有约2.5mg重组的Protein A。2 ml Protein A Agarose共可以结合约20mg human IgG。推荐的线性流速(Linear flow rate)为 50-300cm/h。
Protein A Agarose配制在TBS溶液中，2ml中共含有0.5ml Agarose beads。
本Protein A 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
P2006	Protein A 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 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 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 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 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 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 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。
3. 抗体纯化：
a. 准备工作：
(a) 用0.45微米或0.2微米孔径的滤膜过滤所用的溶液。
(b) 所有的溶液必须用超声等方法脱气(degas)。
(c) 选择适当的纯化柱，用适当量的Protein A Agarose装填纯化柱。
(d) 用10-20倍柱体积的TBS洗涤并平衡纯化柱，流速可以用恒流泵控制为1ml/min。如无恒流泵，也可以完全依靠重力洗涤并平衡纯化柱。
b. 抗体纯化：
(a) 把含有待纯化的抗体上样到纯化柱。
(b) 待纯化的抗体过柱后，用10-20倍柱体积的TBS洗涤，以去除未结合和非特异性结合的蛋白。洗涤是否完全可以通过测定280nm的吸光度进行确定。
(c) 洗涤完后，用10ml 50mM glycine， pH2.7作为洗脱液，洗脱结合的抗体。某些抗体和Protein A的结合能力很强，在pH2.7时洗脱效果不太理想，可以使用50mM glycine， pH1.9作为洗脱液。分管收集洗脱下的抗体，根据蛋白浓度或后续的检测效果确定洗脱峰在哪几个收集管中。
c. 纯化柱的再生：
(a) 用10-20倍柱体积的TBS洗涤纯化柱，使纯化柱达到中性的pH。
(b) 用TBS来保存再生的纯化柱。

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