I am working on a protein which is smaller in size (25kDa), curranty my protein is tagged with GFP which in itself is 26kDa in size and we can't say my protein is localizing because of it's natural localization or GFP is taking it along. Please suggest some good small tag which I can use in fixed cell imaging, live cell imaging and for immunoprecipitation in mammalian cells.
Dear Neeraj Wasnik
These conversations might be helpful, have a look:
1. https://www.researchgate.net/post/What_is_the_best_percentage_protein_gel_for_a_16_KDa_protein_sample_using_Western_blot
2. https://www.researchgate.net/post/How_to_detect_small_molecular_weight_proteins_10_11_17_20_KDa_by_western_blot
Kind Regards
Qamar Ul Islam
HA tag could be good, as it is very small, thus should not interfere with your protein activity, and you can easily detect it by western blot, flow cytometry and immunofluorescence. The only problem is in live imaging, for which you need a self-fluorescent tag. In the past, I succesfully used the Keima tag, fused with small peptides, and didn't noticed any mislocalization of the protein.
Use small tags like Myc, Flag or HA in this case. These are 8-10 amino acid long tags and usually do no interfere with the protein localisation. The only issue is you need to perform immunostaining to visualise your protein with these tags as they are not fluorescent like GFP thus they can’t be used for live cell imaging. Plenty of antibodies are available against these tags and thus immunofluprescence should not be a problem
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