Hi everyone!

I'm trying to detect differences in H2AK119Ub expression levels by western blot in human stem cells, but I've had mixed success. While I'm able to detect clean bands, the signal is consistently very weak, regardless of western blot conditions, which makes me wonder if my protein extraction is suboptimal.

So far, I've used RIPA buffer and/or SDS sample buffer to prepare whole-cell lysates, since several papers showed success with using these methods. However, I've also seen studies use nuclear fractionation or acid extraction, which seem to be the "gold standards" for histone preparations.

My questions are:

• Is RIPA buffer or SDS sample buffer sufficient for histone detection, or is the weak signal likely due to improper lysis conditions?
• Would you recommend switching to nuclear fractionation or acid extraction for more reliable and reproducible results in future experiments?
• Are there specific western blot tips for improving detection of H2AK119Ub in particular? The protocol I follow is quite standard but I'm happy to share more details if needed

Thank you very much for any insights!