Immunoprecipitation Of Proteins
SP, Jan 2005
The immunoprecipitation technique involves isolation of a molecule (usually protein) using a specific antibody that is or can be bound to a solid matrix. Co-immunoprecipitation of a protein involves using an antibody specific to another protein that the first protein can bind to. In general, immunoprecipitation involves these steps -
I. Antigen solubilization
The source of protein of interest may be liquid, such as cell culture conditioned medium, in which case one need only spin down the medium to remove cells and debris before proceeding further.
One needs to prepare cell lysates if using cells as the source of the protein. Lysis can be done using detergents such as Triton X 100 or by mechanical means such as passing 15-20 times through a 25G needle. If using denaturing ionic detergents such as SDS (usually 1%) or Sarkosyl, remember to add 9 volumes of similar lysis buffer, but with a non-ionic detergents such as Triton or NP-40 (usually 1%), to the lysate before proceeding further. Also, the denaturing lysis buffer should have 5 mM EDTA and 15 U/ml DNAse I.
Protease inhibitors should be added to the lysate or medium. Cocktails are best but 1 mM PMSF and 2 ug/ml leupeptin together can suffice.
II. Clearing of lysate
Preclearing helps to remove non-specific proteins that bind to protein A or G beads. 15-20 ul solid beads per ml of lysate are incubated with the lysate with mixing at 4 deg for 30 min and then the beads are removed and discarded following a microspin. This step is optional but required when working with material that have a lot of unrequired protein A or G-binding proteins (such as spleen or thymus lysates - have endogenous immunoglobulins).
Preclearing may be essential if you are using cell culture-conditioned medium (containing serum), ascites fluid, serum, etc.
Note that each ml of protein A or G conjugated to agarose has capacity to bind 10-20 mg IgG. Fetal bovine serum has about 0.2 mg /ml IgG - values for ascitic fluid and high-titer antisera are 5-20 mg /ml.
This step involves setting the reaction so that the antibody binds to the antigen and a solid matrix binds to the antibody. If one has the antibody conjugated to the matrix (usually protein A or G covalently linked on agarose microbeads), it is added directly to the lysate or medium that has the protein of interest. Else, the antibody is added and allowed to bind to the protein and then the matrix is added. All steps are at 4 deg with rotation of reaction tubes for adequate mixing.
A typical immunoprecipitation reaction has this composition - 1-1.5% non-ionic detergent, 10-50 mM Tris buffer at pH 7.4 and 140 mM sodium chloride (or PBS) and protease inhibitors as detailed above. Thus, one can add appropriate chemicals and water to lysates prepared in buffers with other compositions. Final SDS concentration should not exceed 0.1%.
Antibody amounts to use will depend on the levels of target protein, efficiency of the antibody, etc.
1-2 h after addition of antibody, protein A or G beads are added to the reaction. After another 2-3 h (or overnight), most of the target protein would have precipitated. 5-20 ul of the beads are enough for 200 ul reaction volume.
If the antibody is very dilute (e.g., unconcentrated hybridoma culture medium), one may incubate the protein A/G beads with the antibody first (3-6 h to overnight at 4 deg C) and then use spun-down beads (need not be washed).
IV. Washes and further processing
Washes should be done over atleast 5-10 minutes to allow non-specific proteins to diffuse from the interior of beads. Ideally it should be done at 4 deg over 30 minutes, with each wash lasting 5 minutes. Beads are easily pelleted at 100-200 g during a 30 sec - 1 min spin.
Ordinary PBS or TBS, or those modified for higher salt concentration (upto 500 mM sodium chloride; usally 300 mM), and with 0.1% detergent is a good wash buffer. Note, however, that the strength of protein interactions vary from case to case, and high salt concentrations can break interactions. It is good to store such wash buffers at 4 deg with 0.02% sodium azide. Wash atleast thrice, with final wash done in PBS or TBS without high salt or detergent (for good migration on PAGE, etc.). Washes are done in the same tube as immunoprecipitation, using 1 ml buffer each time. After spins, supernantant is carefully aspirated (leaving 10-15 ul with the beads).
The beads can then be stored at -20 or -80 deg, or boiled in SDS-PAGE buffer for electrophoresis or their bound protein eluted using, for example, a low pH (2.5) glycine buffer.
Some prefer allowing the antibody to conjugate to the beads before they are added to the antigen colution. Ths involves incubating the antibody with the beads in the immunoprecipitation buffer for an hour or two and then pelleting the beads. This way, there are no soluble antibodies in the immunoprecipitation reaction to lower the efficiency of antigen binding to the antibody-coated beads, nor are there non-specific proteins to compete with the antibody in binding to the beads.
Protein A or G beads have efficiency of binding 10-20 ug protein per ul.
Most antibodies bind better or equally well to protein G than A - however, protein G binds only IgG antibodies.
Human IgG2 and rat IgG 2a and 2b, and chicken and donkey antibodies do not bind to protein A. Chicken antibodies do not bind to protein G either.
Unlike protein A or G that bind to Fc domain of antibodies (on heavy chains), protein L binds to kappa light chains (all antibody isotypes).
To eliminate or reduce signals from the antibody used for IP when using a secondary antibody in Western blots, etc., (this happens because the light chain of the antibody is bound to the heavy chain by disulfide linkages, and, unlike the heavy chain, is not directly conjugated to the beads; also, some heavy chain itself may come off the beads), one can use a primary antibody of a different isotype or from a different species. One can also, heat the beads in SDS-PAGE buffer without reducing agent for 15 minutes at 65 deg C to collect the supernatant containing bound proteins. Reducing agent is then added to the supernatant before boiling it prior to PAGE.