Cell lysis from tissue or cell culture is straightforward for Western blotting applications in which harsh lysis conditions (e.g., an SDS-sample loading buffer) will not interfere with the results, because the sample will be denatured during procedure. For an IP/co-IP, the lysis should be relatively mild so as not to affect the antibody-antigen binding, but harsh enough to efficiently extract proteins from the cells. It may be especially challenging to find the perfect compromise for membrane proteins.
No immunomagnetic beads is totally inert, and the more complex the sample, the more background binding can occur.
The pre-clearing step will reduce the background that is due to adhesion of sample components to the magnetic beads. This step should not be done without very good reason. Because，pre-clearing will disturb binding between antibody and antigen.
The idea of the binding step is to form the ternary complex of immunomagnetic beads and antigen (protein).
The buffers used in this step (as well as in the washing step) are crucial elements for success. The order of addition of these three components can also be critical. Antibodies can be pre-bound to magnetic beads and the lysate can then be added to the immobilized antibody.
Alternatively, the (non-immobilized) antibody can be incubated with the lysate to form the antibody-antigen complex in solution. Next, the Immobilized Protein A or G immunomagnetic beads is added to purify the antibody-antigen complex from the mixture. You can compare both protocols to see which provides the best results in your particular immunoprecipitation system.
Ideally, washing will delete all nonspecific interactions while preserving the specific interaction between antibody and antigen (and antigen and binding partners for co-IP).Washing with additional Lysis Buffer is useful, as it contains mild denaturants that can help break nonspecific interactions.
If background is a problem, increasing the stringency of the wash buffer may help obtain a more purified antigen and antigen complex from the sample.
Usually accomplished with very harsh conditions such as boiling of the beads in a Reducing SDS Sample Loading Buffer. When using immobilized antibodies, milder elution (pH-shift) are applied to avoid disturbance of the antibody. In this case, only antigen (antigen and binding partners for co-IP) are eluted free of antibody contamination.
Typically detected by Western blotting. In co-IP experiments often very small amounts of protein are available for detection, so high-sensitivity Western blotting substrates(such as ECL) are required.
Lysate in IP-Buffer — positive control confirms sample contains protein of interest. If not detectable, your sample may not contain the protein of interest. However, consider dimerization / multimerization of protein if bands are not visible at the size of the monomer.
IP Flow-Through - confirms whether antigen or binding partners bound to immunomagnetic beads. 1st Wash Step - provides information on whether Wash Buffer is excessively stringent.
Post-Elution Bead-Boil -boiling beads following elution in Reducing SDS-Sample Loading Buffer to confirm efficiency of the elution.
Several researchers have suggested how a mild in vivo crosslinking treatment can provide superior results in ChIP experiments.
Such as, photo-amino acids, which are zero-length linkers, can provide an excellent way to capture the binding before starting a co-IP.
A label-transfer membrane method is a valuable alternative or can act as a confirmation method for co-IP, when it is possible to use a (recombinant) bait protein, instead of an antibody, to bind the prey proteins.
Blocking of Protein A and Protein G immunomagnetic beads is often performed with 1-4% BSA for 1 hour (or 1-5% fish gelatin) at RT or 4°C.
Methods to effectively block the Protein A /G/L immunomagnetic beads without introduction of foreign proteins (which might interfere with later analysis) are outlined below:
Protein A immunomagnetic beads: 50 mM Tris, 200 mM glycine, 1% Tween®-20, pH 10,6 treated overnight at RT
Protein G immunomagnetic beads: 50 mM Tris, 200 mM ethanolamine, 1% Tween-20, pH 10,6 treated overnight at 4°C
Protein L immunomagnetic beads: 50 mM Tris, 200 mM ethanolamine, 1% Tween-20, pH 10,6 treated overnight at 4°C