this is great, but i found after crosslinking, the binding between flag tag and beads are much weaker. is this normal, how to avoid this

Thank you for trying our protocol.

In our experiments, DSP cross linking did not affect IP efficiency so much. However, I think it depends on the protein of interests (POIs), and it is possible that DSP treatment affects IP efficiency. One possibility I can think of is that your POI is included in a huge protein complex, and the cross linking makes it difficult for the FLAG-antibody to access the FLAG-tag fused to your POI. The binding affinity between your POI and the interacting proteins might not be so strong since your IP was successful without DSP cross linking.

To overcome such a problem, I would do...

1. Change the location of the tag (e.g., N terminus to C terminus). It might expose the FLAG tag out of the protein complex.

2. Adjust (lower) the DSP concentration to prevent huge protein complex formation.

Increasing the sample size is another way to get sufficient amount of POI-interacting proteins for the next experiment such as mass spectrometry.

Also, make sure you add reducing agent such as 2-ME to the samples for western blotting. Without reduction step, the protein complex including POI sometimes doesn't migrate into the gel and accumulates on the surface of the gel if the crosslinking is too much.

I'm sorry for the late reply.

I hope this helps your experiment!