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Transfection and Western - (Oct/15/2001 )

I have been trying very hard to transfer cos-1 cells with a construct cloned into pHM6 vector and then detect the expressed protein with anti-HA antibody.But I never detected my protein although pHM6-lacz positive control worked OK. Is that possible that the protein was lost during lysis? What lyzing buffer is good for this purpose? Also, When I did Western detection, non-specific cellular proteins always gave strong signal. How can I eliminate these anoying bands?Your help will be greatly appreciated!Huating


Hi! Have a nice day.You could try to change another anti-HA antibody and repeat your experiment.


Thank you very much for the suggestion. Actually, I just got it work by optimizing a few steps. Now, the problem is the detected protein level is very low. Do you have any idea how to improve the transfection efficiency and protein expression level?