adding C terminal tag to reverse primer (View forum version)



sara.r

Posted 25 October 2009 - 12:01 AM

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

fishdoc

Posted 25 October 2009 - 04:51 AM

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




Tags can interfere with activity. I don't know if it's a common event, but any time you're adding something extra to a protein, there's a chance it affects the structure of it.

I don't know of any C-terminal preferences. I'm currently doing some stuff with Flag, but still trying to work out some optimization, and haven't gotten to actually looking at if it works very well.

A hydrophilic spacer is sometimes used to ensure the epitope tag is available and not contain in a hydrophobic domain within the protein structure. However, if you're going to be doing western blots, and you use denaturing conditions, a spacer shouldn't be necessary, because your protein will be denatured.