Usually, for IP, it is better to use tags like HA, Flag.

293 is extremely easy to transfect, you may double check the plasmid is correct, or have another transfection reagent to try.

RIPA is good for western, CST #9803 is better for IP.

Before running the IP and WB, have you double-checked the following steps? 1) The cloned construct size (vector plus the insert; pc-DNA 3.1 plus PD-L1) by PCR. Also, I would sequence the cloned construct. 2) whether the transfection was successful. As there are several steps in WB, even one that goes wrong could prevent you from getting the right protein band. It will be easier to figure out why you did not get the protein expression (the appropriate band in WB) if you share the protocol (all the steps) you have carried out from the start to the end.

I agree with all the comments above. Do you have problems with detecing PD-L1 during immunoprecipitation? In this case, one reason could be that RIPA buffer is too strong and already distores any existing binding activities between your proteins of interest during immunoprecipitation?

Maybe lysis buffers based on NP-40 or Triton X-100 would be better suited for your experiments?

Best regards,

Christian

Christian Wechselberger Chandra Somasundaram Feng Wang

Thank you everyone! I have checked cell lysate by His-antibody and PD-L1 antibody. The results showed that His-tag was removed from protein so I can't detect His-PD-L1 by his-antibody in WB experiments.

PD-L1 has a signaling peptide in 1-24 amino acids, so the His-tag before signaling peptide will be removed together in cell. I should put the tag in C-terminal or after signaling peptide.

Thanks agian! Hope my experience will help others.