I'm trying to polarize THP-1 and J774a.1 macrophages into M1 and M2 subtypes. I lyse the polarized cells in RIPA buffer and quantitate proteins using BCA assay. However, I always see stark difference in protein amounts in M1 lysates, despite equal protein loading. Has anyone else seen this in their experiments? If so, how do you normalize protein amounts.
All insights are welcome. Thank you!
Hi there, You've likely found the answer by now but I imagine it is not uncommon to have difference total protein expression between M1 and M2. Do you give them both time to rest (~3 days) after differentiation? Are there substantial differences in cell counts?
Either way, it should not be a huge issue for western blots or similar as you just load less volume to account for protein concentration differences.
Richa Khanna , you mentioned that after polarized THP-1 and J774A.1 macrophages were lysed with RIPA buffer, although the total amount of protein measured by BCA was the same, the protein expression obtained by Western blot was significantly different.
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The answer to this question comes from MedChemExpress (MCE) Technical Support.
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