免疫共沉淀(Co-Immunoprecipitation)原理及实验方法-英文

Principle:
Co-Immunoprecipitation (Co-IP) was developed from the immunoprecipitation technique with which Co-IP shares the fundamental principle of the specific antigen-antiody reaction. Co-IP helps determine whether two proteins interact or not in physiological conditions in vitro. Graphically, the Co-IP principle is as described in the right hand side picture.
The known protein (antigen) is termed the bait protein, and the protein it interacts with is called the prey protein. The standard Co-IP protocol is the same as that described for IP, and actually any system designed for IP should also work for Co-IP.
After that cells are completely lysed under non-denaturing conditions, proteins that bound together are kept. Therefore if you use anti-X to precipitate protein X through Co-IP, then you can get other proteins that interact with protein X
in situ.
Co-IP is applied to test whether two known proteins bind each other in cells, or to find a new protein that interacts with a known protein.
Advantages:
1.Proteins that interact in a typical Co-IP are post-translationally modified
and conformationally natural.
2.In Co-IP proteins interact in a non-denaturing condition which is almost
physiological.
Disadvantages:
1.The signals of low-affinity of protein interactions might not be detected.
2.There might be a third protein in certain protein-protein interaction.
3.To choose an appropriate antibody, the target protein needs to be
properly predicted. Or there would not be a positive result in Co-IP.
Reagents and buffers:
•PBS
•RIPA (RadioImmunoPrecipitation Assay) Lysis buffer:
Tris-HCl: 50 mM, pH 7.4
Nonidet P-40 (NP-40): 1%
Deoxycholate Na：0.25%
NaCl: 120 mM
EDTA: 1 mM
*PMSF: 1 mM
*Leupeptin 1 μg/ml
*Aprotinin 1 μg/ml
*Pepstatin1 μg/ml
*Na3VO4: 1 mM
*NaF: 1 mM
Note: Ingredients labeled with * should be added right before each use. PMSF degrades to a half after 30min in water.
•Washing buffer :
lysis buffer:5M NaCl =100:1 (ensure NaCl not exceed 1M)•protein A/G-agarose beads
•Specific antibody (MAb or PAb)
Protocol:
DAY 1
1.Carefully wash cultured cells with pre-chilled PBS for 2 times.
2.Add in cold RIPA lysis buffer (1ml for 107cells).
3.Scrap cells off to clean 1.5ml eppendorf tubes with a clean, cold scraper.
Put them on a low-speed rotating shaker for 15 min at 4°C.
4.Centrifuge at 14,000 g 4°C for 15min, transfer the supernatant to new
tubes immediately.
5.Wash protein A/G-agarose beads for 2 times with PBS and make a 50%
protein A/G agarose working solution (in PBS)
6.Add in 50% protein A/G agarose with ratio of 100μl for a 1ml sample
solution. Shake on horizontal shaker for 10min, 4°C (This step aims to eliminate non-specific binding proteins)
7.Centrifuge 14,000g at 4°C for 15min, transfer the supernatant to new
tubes and discard protein A/G-agraose beads
8.Quantify total protein with BCA assay or other methods.
9.Dilute the total protein to 1μg/μl with PBS to decline the concentrations of
detergents. If you feel the concentration of your target protein is low, you can dilute the total protein to 10μg/μl. (if it’s high enough)
10.Add in appropriate amount of primary antibody to approximately 500μl
total volume..
11.Slowly shake antigen-antibody complex on rotating shaker at 4°C for
overnight.
Note: if downstream experiment is enzyme activity assay for kinase or phosphatase, it’s better to change step 11 to a 2h incubation at room temperature.
DAY 2
12. Centrifuge 14,000g for 5s, keep the pellet and wash with pre-chilled
w ashing buffer (or cold PBS) for 3 times. (800μl each)
13. Collect the supernatant to proceed to SDS-PAGE, western-blot, or mass spectra analysis.
Note: This Co-IP protocol is to bind antibody to the Protein A/G-argarose beads and then mix with the antigen. It gives lesser yield than the other one and avoids the problem of co-elution of antibodies. If you want to yield high purity of target protein regardless of non-specific binding, you can mix antibody with protein sample prior to addition of Protein A/G-agarose beads, thus in the end the antibodies are also co-eluted with target protein and interference might occurs in western blot detection.。