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Molecular cloning using an ezh2 insert

molecular biology cloning vector insert his-tag

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#1 SarahS_

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Posted 27 November 2014 - 08:56 AM

Hi! :)

 

I am confused about his-tags and the way they work. If i am cloning the EZH2 gene in a vector/plasmid, why does the his tag have to be sited on the amino terminal of the ezh2 protein?

 

Thank you!



#2 bob1

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Posted 27 November 2014 - 09:33 AM

Basically there are two reasons why you might need to tag at the N-terminus...and one of those isn't really a reason, just a convenience:

 

One scenario is that tagging at the C-terminus causes the protein to misbehave in some fashion (doesn't transport properly, doesn't fold properly, loses some interaction, etc.), this is very valid reason for concern and definitely a case where you would avoid the C-terminus. The other scenario is that the plasmid you are inserting it into is a N-terminal tagging plasmid -the tag is already in the plasmid and upstream of the multiple cloning site, so you don't have to amplify the insert with a His tag in your primers.



#3 SarahS_

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Posted 27 November 2014 - 11:51 AM

Basically there are two reasons why you might need to tag at the N-terminus...and one of those isn't really a reason, just a convenience:

 

One scenario is that tagging at the C-terminus causes the protein to misbehave in some fashion (doesn't transport properly, doesn't fold properly, loses some interaction, etc.), this is very valid reason for concern and definitely a case where you would avoid the C-terminus. The other scenario is that the plasmid you are inserting it into is a N-terminal tagging plasmid -the tag is already in the plasmid and upstream of the multiple cloning site, so you don't have to amplify the insert with a His tag in your primers.

 

Perfect! Thank you so much!







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