【求助】免疫共沉淀试剂选择

最新回复

• birdfish (2013-12-07 21:07:02)

  
  单独买protein A或者G beads就可以了，这个最好买进口的，santa Cruz的便宜也不贵的。
  其它试剂，其实就是裂解buffer，最好自己配制，买kit反而不好，因为不同的实验要求的去垢剂成分不一样，盐离子浓度也不一样。

• koook5695 (2013-12-07 21:07:20)

  
  嗯，谢谢！先试试看。不过现在又遇到一个新的问题：做CO-IP的时候转染细胞是在六孔板进行的吗？这样收的蛋白不是很少，到时候加蛋白A/G的时候就不知道怎么加了，看PROTOCOL都是说1ml-3ML加多少试剂！

• birdfish (2013-12-07 21:07:40)

  一般内源蛋白coIP至少要一个10cm dish，也就够做2-3次，如果抗体不是很好的话。过表达的一般6cm的够了。
  10cm dish，1ml裂解液，30ul饱和的beads吧

• huifeng0516 (2013-12-07 21:09:47)

  上海生工有
  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℃.

• mysmdbl (2013-12-07 21:10:10)

  
  蛋白A或G的话你要和你的抗体相吻合。不推荐买蛋白A。它的结合谱窄一些。

• 30moonriver (2013-12-07 21:10:29)

  
  好的 知道了！买的是蛋白G，做了一次结果还好！可是又发现这个怎么定量呀，因为我有一个是突变体，想看看突变体对于结合有没有什么影响，所以要定量，我现在没想到怎么定量和怎么用内参！