I have GFP tagged beta globin cloned in pcDNA3.1. After doing lipofectamine or electroporation mediated transfection, I am able to visualize the fluorescence imparted by GFP. But the western blot is not working even after taking as much as 100 ug of protein lysate. I have used both beta globin and GFP specific antibodies.
I suggest trying a positive control, you need to determine if it's your antibody or your sample. Are there control lysates available, or do you have any cell lines in house that should endogenously express beta-globin? That's a good start for troubleshooting. Can you see any housekeeping genes on your western blot? GAPDH or B-Actin?
I agree with Craig that having positive controls will help you rule out any procedural errors. Otherwise, are you sure that your promoter functions to also translate beta globulin? I'm sure that's a trivial yes, so you might also want to just increase your transfection efficiency (e.g. with optimized reagents for HEK293: https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/) and that could help amplify a potentially faint signal.
Thanks for the suggestions.
As mentioned, the antibody works fine in the controls. I am able to see the overexpression of beta globin tagged with GFP by fluorescence microscopy. But the western blot is not working.Also i have tried both semi dry and wet transfer method.
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