how to visualize a peptide - (May/20/2010 )

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We haven't done that yet. We have a precursor of this peptide, a longer fragment from the holoprotein, with gfp tagged. But I can't see anything green by overexpressing this tagged protein fragment. I'm afraid the gfp tag won't work for my interested peptide as well.

medchemgirl on May 26 2010, 07:22 AM said:

As I understand, u are just expressing a small peptide, so it's not a protein perse, is it just a fragment of a protein? Why don't you express the whole thing tagged to GFP? you should definitely see the GFP construct. Make sure you are not fusing the antisense, otherwise you won't get it, and it has to be in frame. Check your sequence.

-proteinrunner-

First, have you done the control transfection of the GFP only so that you know your transfection, cells and DNA are good? If you can see the control GFP but not your GFP-tagged peptide, then something is wrong with the DNA. Is your peptide tagged on the NT or CT? If it's the CT, I'd be concerned that the GFP is not in the correct frame or that you've got a mutation that created a stop codon early on in the GFP. Also, make sure you have a good Kozak sequence. Recheck the sequence carefully!! The other idea I have is that for some reason that I can not explain, I have one construct in particular that the transfection or expression (not sure which) declines rapidly with each and every freeze/thaw. It is the only construct that has this particularity and after about two freeze thaws, it's completely dead. Just try to do a quick miniprep and make a fresh DNA prep before the transfection.

-rkay447-

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