Confirm that the Western blot works well. Appropriate positive and negative controls should be used, including a whole-cell lysate as positive control or use our positive control (supplied in KIT for tagged protein). Then check the following:
The lysis step
Perform all steps at 4°C to reduce proteolysis, dephosphorylation and denaturation. This is especially important for the binding step which is typically incubated overnight (or at least 2 hours) at 4°C.
When working with phosphoproteins also need to add phosphatase Inhibitors.
Try different lysis buffers (harsher or less harsh) and, as mentioned above, use a whole-cell lysate as positive control to confirm that protein extraction works (especially important when working with membrane proteins). The least stringent lysis buffer that gives an acceptable yield of the desired protein can be considered "optimal".
For co-IPs, use only gentle vortex steps and wide pipette tips. Be very careful not to disrupt any protein complexes. In addition, if any additives are required for the proteins to interact, these should be included in the binding buffer to enhance capture of the protein complex.
The binding step:
Avoid reducing agents because they can cleave the antibody.
Use the "classic" IP method without covalent antibody immobilization on beads. The immobilization can reduce the antibody's affinity to the antigen and prevent IP. This is more likely to happen with monoclonal than with polyclonal antibodies. Direct immobilization of the antibody to magnetic beads typically results in less functional loss to the antibody than does crosslinking the antibody to Protein A or G, but may still reduce the ability of the antibody to bind its antigen.
Here are some advices for you:
1. Avoid the pre-clearing step referred to in Additional Considerations, Pre-clearing, above.
2. Increase the amount of antibody and/or sample (e.g., incubate lysate aliquots sequentially until a sufficient amount of protein has come in contact to the beads).
3. To keep this practical, incubation of the binding step is typically done for 2 hours at 4°C.
4. Do not use samples that are very highly concentrated because they are more likely to encounter background problems.
5. Over-express your bait protein, being careful that expression levels are not so high that the protein cannot be properly folded in the cells, resulting in artifacts or loss of binding.
6. Try different binding conditions. Refer to Additional Considerations, IP Buffers above (e.g., buffers with different concentrations of detergents or different detergents, especially when working with membrane proteins).
7. Try in vivo crosslinking (to stabilize protein complexes in case of co-IPs) as mentioned in Additional Considerations, In-vivo Crosslinking.
The washing step:
Use less stringent washes If necessary, cut down on the number of washing steps.
The elution step
If elution was done with a pH-shift, also need to check if boiling the beads in SDS-sample loading buffer results in a higher protein yield. However, background will rise as antibody fragments will appear in the eluate.
The detection step:
Increase the sensitivity using one of the following options:
Metabolic labeling of proteins (e.g., by radioactive or heavy amino acids – the latter only for MS based detection methods).