I'm doing WB to detect Gtf2h4 in mice's thymus and spleen. I used 4 different Ab from different companies and no one seems to work, but the positive control (histone 3) was perfect. I tried with different blocking buffers (2 different milks and BSA). I've tried with different secondary Ab concentration (1:1000, 1:20.000 and 1:50.000). Now I'm doing dot blots to see if the problem was the gel, but even the dot blot is not working. I don't have clue about what is going on. Thank you for the answers.
Hello, Virginia.
Suppose you have done standard procedure with proper nuclear extract. Then I would say H3 is one of the most abundant nuclear proteins. On the other hand, Gft2h4 is a luxurious protein that is activated only by certain cellular responses. Probably your activation treatment did not reach over the infection threshold to lead the gene activation, txn and tln. Not strong enough.
Have you already seen transcript in the same tissues or cell culture? Then, there may be translational regulation(s) may be present.
Regards.
Thank you a lot for the answers! I've tried with the nuclear protein fraction of a concentratio of 7x106 HEK and it worked, so now I'm trying with higher cellular concentration of WT splenocytes for the nucler proteins extraction and I hope it will work!
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