We are trying to detect and quantify the presence of KLF2 which should appear as we have memory cells (CCR7+/CD62L+). We're using western blotting for that and we've tried both the traditional "long" method, and we've tried ProteinSimple's Wes Machine. Neither can produce a confirming band.
-We've tried multiple antibodies from different companies
-We've tried checking our cultured cells from D0 through D6 every day to see if KLF2 may have come and went
-Maybe we thought that KLF2 exists in small quantities, so to exclude the noise from extracting all the proteins, we've done a nuclear extraction and ran a western blot on that- No band
- We've tried varying both input protein concentration and antibody concentration
Any suggestions would be great! We've been trying to detect this elusive KLF2 for a while.
We've ran western blots on many proteins, and those work fine. Only KLF2 doesn't work for some reason...
I would first go for a good positive control! Try and get hold of some adipose, stomach or lung tissue, which should contain KLF2 and run the western blot. Also, try and include an immunoprecipitation, and co-run a qPCR on your memory cells at the same time. Hope it works out for you!
I am not an expert in this particular target, but it seems that proteasome inhibitor Velcade is boosting levels of KLF2.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4055929/
You can try to boost KLF2 levels by treating your cells with Velcade, or at least treat them with protease inhibitor cocktails (cOmplete) during protein isolation to ensure integrity of KLF2.
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