I was did recombineering to express membrane localized protein into the bacterial chromosomes using double GFP to increase the fluorescence signal Intensity . I got good number of recombinat colonies after efficient transformation . I was tested the ON growth recombinat colonies by Fluorescence Microscope and got positive florescence signals . The size of inserted gene into chromosome also confirmed by colony PCR but the location of expressed protein was not as I expected . The protein is membrane localized and I am expecting even distribution on the membrane but what I got is 2-3 localized protein on the membrane and it looks like Inclusion bodies . Here all my experimental procedures was correct and but the signals is not improved and the location is not as I expected . Anyone rich experience in recombineering .
Hi there,
So genetically your experiment works but it is not giving you the expected protein localization signal, right?
So have you checked the clones for their content in GFP containing proteins? If everything is OK you should obtain a single band in WB using anti-GFP antibodies with apparent MW of the fusion protein.
If fusion is actually present it might be that GFP has an effect on the processing of the fusion protein leading to mislocalization (in that case it might be worth trying the same kind of experiment with some smaller tag and therefore less side effect on protein localisation)
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