I have a sequence cloned in pcDNA6 myc/his B vector (with proper Kozak at initial of insert) with myc- and his- tags in proper frame followed by stop codon. The clone sequencing results are desired. I am able to detect over-expression at mRNA levels by qPCR. But when I go for western blotting of the same, I can't detect any expression. I always get band for positive control suggesting that my western protocol is fine. What can be the probable reasons for this problem.
Please help.
I lyse the transfected HEK293t cells by NP40 buffer+protease inhibitor. The proteins I am dealing with are cytoplasmic and not membrane proteins as per literature.
Is there a chance your mRNA is the target of a cellular miRNA? Can't you run the sequence through something like TargetScan (http://www.targetscan.org/vert_71/)?
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