Ensure fresh protease inhibitors are added when cells or tissue is lysed.
Target protein may not be expressed in sample used or low level of target protein expression in the sample used.
Check the expression profile of the target protein to ensure it will be expressed in the cells of your samples. If there is low level of target protein expression, increase the amount of lysate used. However, this may lead to increased non-specific binding so we suggest to pre-clear the lysate before IP procedure.
Check that the recommended amount of antibody is being used. The concentration of antibody may require increasing for optimization of results.
Ensure you are using the correct elution buffer and that it is at the correct strength and pH for elution of target protein.
Ensure you are using the correct immunomagnetic beads for the antibody isotype used..
Check the IP antibody datasheet to see whether the antibody detects denatured or native protein and ensure the correct lysis buffer is used.
a. Verify binding/specificity of your antibody to your antigen, e.g., by ELISA.
b. Check the binding of your IP antibodies conjugated to the beads. If the antibodies do not captured or bound to the antigen, the immunoprecipitation experiment will not work.
c. If you have used the indirect method, try the direct method. Conversely, if you have used the direct method, try the indirect method.
d. Check the amount of beads and sample volume. With reference to the capacity of different beads proposed in the datasheet, increase the amount of beads or the concentration of your antibody during IP procedure.
e.Increase the incubation time.
f. Try another antibody.
The binding sites of your antibody have likely been altered by the crosslinking. When this occurs your antibody will show reduced affinity or no affinity to its target antigen.
This is always a high risk with crosslinking, and it is a problem easily avoided by choosing the Sino Biological primary antibody conjugated immunomagnetic beads.
Another consequence of crosslinking can also be increased affinity for unintended (non-specific) targets.
If you are using reducing agents in the sample buffer before gel loading, try a sample buffer without reducing agents.
Reducing agents such as DTT or β-mercaptoethanol will break disulfide bridges within antibodies resulting in release of the antibody light and heavy chains.
Another option is to elute the protein by lowering the pH or by the polypeptide solution, this will show non-antibody band in gel.