About immune precipitation, here is a detailed description!

Immunoprecipitation (IP) is one of the methods to use antigen antibody specific reaction to purify enriched protein.

As a technology to study the role of proteins in natural state, IP is widely used in scientific research.

Enrichment, or it can be used to separate the natural state of protein or the interaction of protein complexes (such as immune coprecipitation Co - IP), can also study the interaction between proteins and chromatin DNA (ChIP), and so on.

• The principle of article

  
  

Antibody and cell lysis solution or protein expression in the supernatant after the corresponding protein, protein A/G again (ProteinA/G) (or resistance) coupling agarose or incubation Sepharose beads and beads - protein A/G is obtained by centrifugal (or resistance) - antibody - protein complexes.

Complex precipitation after centrifugal washing sample weight hanging on the electrophoresis buffer, boiling water bath 5-10 min, in under the action of high temperature and reducing agent, dissociative antigen and antibody, centrifugal supernatant collection, included in the supernatant antibody (light chain and heavy chain has broken) and the target protein.

The mixture was separated by SDS - PAGE electrophoresis and Western blotting development, and finally determined whether the immune precipitation experiment was successful based on whether there was a target protein strip on the film after the development.

2.Process essay

First，The preparation of Lysate

Then，Immunoprecipitation (capturing target protein)

（1）1-3 mg lysate add 3-5 ug a resistance, 4 ℃ rotation for the night, form "& target protein antibody" dimers;

（2）Add 50 ul approved by PBS or incubation liquid processed proteinA/G beads, 4 ℃ rotating 1-4 h, forming "- target protein antibody - proteinA/G" trimer;

（3）Washing liquid detergent 5 times, remove protein;

（4）Ph 2.0 pickling, fully eluting the antibody and target protein.

The fourth step can also be boiled, namely: add the sample loading buffer (final concentration 1 X) to boil 5 ~ 10 min, and 12, 000 RPM centrifuge for 3 min.

The last，WB test

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