Detailed description of the immune precipitation process
1.Lysate preparation
（1）Observe the cold operation throughout the whole process: PBS for cell collection is pre-cooled on ice, and the slurry ice operation is organized.
（2）Liquid nitrogen grinding vs. glass homogenizing: the organization is given priority to use liquid nitrogen grinding;
Organize less use of ice bath glass homogenizer.
（3）Lysis liquid is commonly used in RIPA: a certain amount of protease inhibitors, such as phenyl-sulfonyl fluoride (PMSF), Na3VO4, etc.
（4）Note: the cell is usually 180 W ultrasonic 1 ~ 2 min (2 s);
The tissue can be 180 W ultrasonic 2 ~ 5 min (super2 s).
2.Objective protein enrichment
（1）
Choice of acid washing and boiling method: acid eluting is suitable for the washing of all sizes of protein, and the background is cleaner.
The boiling method may boil A small amount of protein A/G on the Beads, which is generally not recommended for the purpose protein of 50 kDa.
（2）The Beads-Protein A & Beads-Protein G Choice basis: look at the table first, we explain again!
Protein A and protein-protein G relative bond strength
The space is variable
+ + + = strong combination
+ + = medium combination
- = Weak or not binding
From the above table, if the rabbit antibody (or mouse IgG2a / 2b / 3) is captured, then it should be used with the Beads - Protein A;
If the IgG1 antibody is captured in mice, then the Beads - Protein G should be used
（3）The single reactance of IgM and IgA is not suitable for IP, on the one hand, because the molecular structure of these two kinds of antibodies is poor, and it is easy to lose the ability to capture antigens in lysis fluid.
On the other hand, because ordinary packing can not effectively capture these two kinds of antibodies in lytic solution, it is also easy to have negative results.
3.WB test
The selection technique of resistance
In addition to the target protein, there are also antibody heavy chains and light chains.
How to avoid antibody heavy/light chain interference when the target protein is close to the antibody heavy chain or light chain?
Here are two tips:
（1）Use opportunely HRP - protein - A second resistance analogues or Mouse anti - Rabbit Light chain of antibody
Compared with conventional sheep anti-rabbit or sheep anti-rat, the use of hrp-protein A diantigens can effectively reduce the combination of anti-body weight chain and the basic elimination of the binding of antibody.
And Mouse anti - Rabbit light chain antibody can effectively eliminate the Rabbit anti the combination of heavy chain in this way, can be combined to choose the two as WB resistance.
On purpose in 45 ~ 55 kDa protein size, use the Mouse anti - Rabbit light chain antibody as WB two resistance, the rest of the size range can choose HRP - protein - A second analogues.
（2）The antibody and WB were detected and the antibody was selected from different species
The antibody and WB were detected by the antibody, and the antibody was used to prevent the interference of the antibody.