cloning
ashraf bakkar
Saturday, 08 November 2008 22:26 UTC
we cloned our gene of interest in PCDNA3.1.
I transfected my cells. I extracted RNA and did RTPCR and results showed that there is a signficant expression of my gene compared to empty vector.
I did western blot using ANTI-V5 ( at the end of my protein) and can’t detect the signal?
what the reason (s) in your opinion?
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Replies
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Do you have a positive control for western blot? First I would make sure the western blot is working, and make sure the sequence is not frame shifted or mutation gaining a stop codon. Then, I would consider biology, for example, whether your protein is processed and cutting the c-terminal.
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Hello,
Did you include a Kozak sequence in your construct? Believe me, it makes a big difference in terms of protein expression.
Good luck.
João
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