Hello!
I am a newbie when it comes to protein purification. I inserted a x6 his tag but when I got my sequencing somehow I ended up getting a x7 his tag. Is it a huge deal that I have an extra histidine? What is the difference?
thanks
margarita
Dear Margarita
i do not think you will observe many differences having one addtional histidine tag in term of purification. Of course this will little change the sequence and biochemical properties of your protein, but also in this case the changes will be little and not so important expecially if in your sequence you have a site for protease digestion and removal of the tag after purification.
In principle, an high number of histidines can guarantee an high number of link beetween tag and metal particle and therefore a stong binding of the protein to the metals. In fact Several scientist expecially for purification of protein extracted for mammalian cells prefer to use the 10Xhis tag instead the 6X his tag.
best regards
Manuele
It shouldn't be any problem, the minimum requirement usually is 6xHis, in some cases people use 10xHis to increase the nickel banding ability. An extra His on either ends will not affect the functionality of your target protein even it is a non-cleavable tag. Your 7xHis tag should have a slight stronger nickel binding ability but practically hard to be noticed.
Perfect! Thank you so much the information. I shall proceed with my experiments;-)
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