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小木虫论坛-学术科研互动平台 » 生物医药区 » 分子生物 » 综合其他 » 【求助/交流】求教做免疫共沉淀的人
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jurkat.1640

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[交流] 【求助/交流】求教做免疫共沉淀的人

我没做过免疫共沉淀，现在要设计免疫共沉淀实验，为了更高的成功率和实验质量，现在请做过的高手简要介绍一下免疫共沉淀最可能遇到的几种问题
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1楼 2010-12-10 16:37:12
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jurkat.1640

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引用回帖:
Originally posted by mourning at 2010-12-10 23:11:23:
问题就是可能有非特异性曝出来

蛋白质的上样量大概多少？
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4楼2010-12-14 16:04:51
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xhjggl

木虫 (正式写手)
★ ★
dhd997(金币+2):热心 2010-12-11 08:42:16
jurkat.1640(金币+3):谢谢参与 2010-12-11 08:58:14
上海生工有试剂盒卖，很好用！
Immunoprecipitation Kit
Components        BS688 For 20 reactions         BS689 For 20 reactions         BS690 For 20 reactions
10x Lysis buffer        10 ml        10 ml        10 ml
PMSF        0.6ml        0.6ml        0.6ml
10x IP        50 ml        50 ml        50 ml
SDS        5 ml        5 ml        5 ml
NaCl        10 ml        10 ml        10 ml
Protein A-Agarose        0.4 ml        /        /
Protein A/G Plus-Agarose        /        0.4 ml        /
Protein A-Sepharose        /        /        0.6 ml
20x PBS        20 ml        20 ml        20 ml
1x SDS gel-loading buffer        1 ml        1ml        1 ml
Spin columns and  tubes         20        20        20
Price        ¥1,520        ¥2,250        ¥1,920
Description:
Immunoprecipitation (IP) is a method by which a protein can be specifically purified from a complex mixture of proteins using a specific antibody and a matrix that binds the antibody. The matrix bound protein (via the specific antibody) can then be separated from the mixture by centrifugation. The matrices commonly used are agarose or sepharose bound Protein A or protein A/G PlUS. IP is one of the most widely used immunochemical techniques. IP followed by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and immunoblotting is routinely used in a variety of applications: to measure the molecular weights of protein antigens, study protein/protein interactions, determine specific enzymatic activity, monitor protein post-translational modifications and determine the presence and quantity of proteins. The IP technique also enables the detection of rare proteins, which otherwise would be difficult to detect, since immunoprecipitation can concentrate them as much as 10,000-fold..
Applications:The Protein-A Immunoprecipitation Kit is especially designed to allow maximal recovery of immunoprecipitates. The whole process is performed in mini-spin columns, instead of in microcentrifuge tubes, which enables convenient washing of the antigen-antibody-bound beads.
Features:Easy access.;Minimal loss of antigen-antibody bound beads during washing.；Minimal non-specific signals by increasing the stringency of the washing steps.
Storage Condition:Stored at 4℃.and PMSF should be stored at -20℃.
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2楼2010-12-10 18:51:57
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mourning

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jurkat.1640(金币+2):谢谢 2010-12-11 08:58:58
问题就是可能有非特异性曝出来
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3楼2010-12-10 23:11:23
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vetzhangshen

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★
小木虫(金币+0.5):给个红包，谢谢回帖
啊怎么没有人回复啦
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5楼2012-02-22 10:54:29
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