Recently we performed expression and extraction of Nef proteins from Bl21 strain (His tag-Nef and Nef-GST), when we run these proteins on sds-page, we observed that His-Nef stay higher than expected weight, (Nef molecular weight is 27 kDa) and His-Nef stay on about 30 kDa. but in fusion with GST(26kDa) it stays about 53kDa (means GST+Nef: 53). Against, simultaneous another protein with same wight (27kDa) in fusion with His-Tag, on SDS page exactly placed on 27kDa. But I don’t know why and How His-tag can affect on Nef structure that it stays higher than expected level. Any one can guide me?
Hi there,
The impact of Histag on protein mobility is very little as MW of the tag is less than 1kDa. Actually some proteins are naturally running higher MW on SDS/PAGE. Did you compare the mobility of His tagged Nef with the one of non tagged protein? As SDS/PAGE is a denaturing technique, any impact of the tag on the protein structure should not be noticable on the protein mobility.
Hi there,
The impact of Histag on protein mobility is very little as MW of the tag is less than 1kDa. Actually some proteins are naturally running higher MW on SDS/PAGE. Did you compare the mobility of His tagged Nef with the one of non tagged protein? As SDS/PAGE is a denaturing technique, any impact of the tag on the protein structure should not be noticable on the protein mobility.
Hi,
which vector are you using? in pET30 for example there is N-terminal Histag and additional sequences for thrombin, S-tag and enterokoinase on N-terminal end and optional C-terminal histag ...so you have to count these too
HIS tag can have 6-10 histidines and trmobin or other enzyme cleavage site that can increase its mass up to 2 kDa
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