My lab is having trouble detecting a lysosomal enzyme with its dedicated antibodies in immunohistochemistry, so we want to try fusing it to an epitope tag to facilitate better detection.
We have decided on two fusions, one with a C-terminal V5-His sequence and one with either 3x c-Myc or c-Myc-AU1. These will be attached to the protein via a linker we have already verified for a different protein.
My question is: what, if any spacer sequences are necessary to separate the V5 and His tags, or the c-Myc and AU1 tags?
Looking at sequences already out there, it seems some people use no spacers at all, some use a single amino acid (eg Glycine), and some use a sequence of five or so. There doesn't appear to be any general rule and almost nobody discusses if/why different sequences are better than others.
I was wondering if anyone had any insights into how to decide on a spacer?
Hi Cook,
After checking with commercial plasmids and constructions in our past works, there were found 0-10 amino acids (0-30 nucleotides) for separating two epitope tags. If you still worry about the shield effect. Try to insert 2 amino acids (6 nucleotides) between two tags first.
But the major point is that the design makes your tags detectable with anti-tag antibodies.
And always keep in mind, the spacer sequence you inserted will not cause frame shift of your constructs.
Good luck.
Hi James - you are right in saying that there is generally no "rhyme nor reason" for the length of spacer sequences.
In the past I have used glycines as the spacers (they are small, neutral amino acids and also allow it to be flexible.
If in doubt place 4-6 glycines in between the tags.
Sorry I can't be more help,
Gary
Hi Gary
Thanks for your answer. I suspected that was the case, so I'll probably go with your suggestion and add in a few glycines.
Thanks again
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