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epitope tagging of pcr products


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#1 sara.r

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Posted 24 October 2009 - 11:57 PM

hi
I am going express some intracellular proteins in mammalian cells and I want to to add an small epitope tag (myc, his. flag,...) to my reverese primer for detedtion of the expressed protein by ELISA or western blotting . I have some questions for this:
1. does adding small C terminal tags would interfere with activity of the protein (I search some commercial vectors in many of them tags are in N terminal).

2. is there any preferance in chosing a tag for c terminal use? I mean if some tags act better when add to c terminal.

3. is it necessary to put some spacer sequences between my protein and tag or I can add it directly to my protein, if yes what kind of spacer I can use.

many thanks

#2 swanny

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Posted 25 October 2009 - 05:18 PM

hi
I am going express some intracellular proteins in mammalian cells and I want to to add an small epitope tag (myc, his. flag,...) to my reverese primer for detedtion of the expressed protein by ELISA or western blotting . I have some questions for this:
1. does adding small C terminal tags would interfere with activity of the protein (I search some commercial vectors in many of them tags are in N terminal).

I don't think so, but you never can tell with proteins!

2. is there any preferance in chosing a tag for c terminal use? I mean if some tags act better when add to c terminal.

A bit of work has gone into this question. look at recent reviews about protein expression systems for high throughput structural genomics consortia for more details; I do know that GST tends to not work well at the C-terminus. We use GFP as a C-tag, with an N-terminal His tag, because NTA columns turn a lovely green as we load the lysate (this shows that both ends of the fusion are present, so the middle will be also!)

3. is it necessary to put some spacer sequences between my protein and tag or I can add it directly to my protein, if yes what kind of spacer I can use.

We have designed our system with TEV- and Precission-cleavable tags. This way the protease sites form an effective linker between the tag and the protein.
All the best with it.
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