Fusion protein expressing problem - (Oct/12/2006 )
Hi everyone,
Good evening! I have a great difficulty recently. I have constructed eukaryotic expression vector pUB6 V5 Hisb contained double fusion genes linked by 10glycines linker. Sequencing seems no mutation in it. Moreover, I also obtained their transfectants in C6 glioma cells. To my depression, never has fusion protein (MW:98KD=48+50)been occurred by western blotting though I have been improving the western blot method:
SDS-PAGE: 7.5% GEL running 65V X 3.5-4hr;
Transfer: 50V X 4hr
AP (BIORAD) color
Result: Nothing in fusion protein; however, my single gene construct appear clear band.
Who can give me any suggestion? Thank you very much in advance!
Good evening! I have a great difficulty recently. I have constructed eukaryotic expression vector pUB6 V5 Hisb contained double fusion genes linked by 10glycines linker. Sequencing seems no mutation in it. Moreover, I also obtained their transfectants in C6 glioma cells. To my depression, never has fusion protein (MW:98KD=48+50)been occurred by western blotting though I have been improving the western blot method:
SDS-PAGE: 7.5% GEL running 65V X 3.5-4hr;
Transfer: 50V X 4hr
AP (BIORAD) color
Result: Nothing in fusion protein; however, my single gene construct appear clear band.
Who can give me any suggestion? Thank you very much in advance!
u must have verified it but, did u check if the sequences of both the genes r in frame.
I have verified it. the same problem is also existed in other fusion construct in previous labmate. according to limited DNA sequencing (especially the C' terminal and linker sequencing), it is no problem. At present, I just readthrough the fusion gene construct.
Actually, the most problem rest with fusion protein stablity because the linker is easy to break. Then, how to effectively solute the possiblity in western blot?
u must have verified it but, did u check if the sequences of both the genes r in frame.
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