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

Hi,

I'm transfecting cells with a construct producing a -10kDa peptide which is very easily degraded. Is there any technique that I can use to visualize the peptide's localization in the cell? I've tried the normal staining but I can't see any thing. I can't even see the WB signal of it.

I hope somebody can answer the question...

Thanks,
Proteinrunner

proteinrunner on May 20 2010, 09:01 AM said:

coexpression with GFP or an analogue to build a fusion protein...

Inmost sun on May 20 2010, 06:53 PM said:

GFP alone is ~27kDa, almost 3 times as the size of proteinrunner's peptide. I think that might cause some problems with the peptide's correct folding and localization.

ProteinWork on May 21 2010, 09:18 AM said:

Not necessarily. GFP is known to increase stability, and certainly solubility of fusion partners.

Where do you predict the protein should be (I think we can call a 10 kDa polypeptide a protein...)?

swanny on May 21 2010, 12:52 AM said:

I think proteinrunner was trying to determine the localization pattern of this protein. I'm just not sure how GFP fusion will affect it. But I don't know any other way to do it, either.

Thank you guys!!

I expect it to be a nuclear protien as a transcription factor. The construct of it has a myc tag, but it's not useful either for WB or IF... We have another longer pepteide (kind of precurcor of the 10kDa peptide) with gfp tag, but we can't see it by IF either...

Any other ideas??

Have a nice day!

If it is myc tagged, you should be able to detect it by antibody - there are lots of Myc antibodies about.

I\ve already tried that, and it didn\t work...

bob1 on May 23 2010, 05:03 PM said:

proteinrunner on May 26 2010, 09:36 AM said:

Maybe you could try to increase the stability of the protein. Using a proteosome inhibitor or something like that.

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.