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Trouble finding 5kDa protein in western blot - (Jul/24/2007 )

I have synthesized a gene which should express the 36 residue villin headpiece with a 10 residue Myc tag fused to the C-terminus. I am trying to verify whether this protein is expressed by western blotting and am having some trouble, I think the problem may be in my detection. The total mass should be 5.3 kDa.

I am running a 15% acrylamide mini-gel in TGS, followed by wet blot in Tris/Glycine/Methanol. The markers transfer well, but unfortunately my smallest marker is 10kDa. I then probe with primary and secondary antibodies and develop, but see no bands where I would expect them.

My questions are:

1. Is there anything wrong with my method? Is there a possibility that the fusion tagging/probing will not work correctly because the villin headpiece protein is so small?

2. Is there any other method you'd recommend I use to check whether my protein is being expressed?

Thanks for any help

-Ra_-

QUOTE (Ra_ @ Jul 24 2007, 03:31 PM)
I have synthesized a gene which should express the 36 residue villin headpiece with a 10 residue Myc tag fused to the C-terminus. I am trying to verify whether this protein is expressed by western blotting and am having some trouble, I think the problem may be in my detection. The total mass should be 5.3 kDa.

I am running a 15% acrylamide mini-gel in TGS, followed by wet blot in Tris/Glycine/Methanol. The markers transfer well, but unfortunately my smallest marker is 10kDa. I then probe with primary and secondary antibodies and develop, but see no bands where I would expect them.

My questions are:

1. Is there anything wrong with my method? Is there a possibility that the fusion tagging/probing will not work correctly because the villin headpiece protein is so small?

2. Is there any other method you'd recommend I use to check whether my protein is being expressed?

Thanks for any help



In 15% acrylamide gel your polypeptide run off. Use 20% gel or make 16% tricine phorez

-circlepoint-

QUOTE (circlepoint @ Jul 24 2007, 10:50 PM)
In 15% acrylamide gel your polypeptide run off. Use 20% gel or make 16% tricine phorez


Would that be true even if the blue dye did not run off the gel? I stopped the electrophoresis right before the bromophenol blue dye ran off the gel, so would the protein be able to run faster than the dye?

-Ra_-

QUOTE (Ra_ @ Jul 25 2007, 10:37 AM)
QUOTE (circlepoint @ Jul 24 2007, 10:50 PM)
In 15% acrylamide gel your polypeptide run off. Use 20% gel or make 16% tricine phorez


Would that be true even if the blue dye did not run off the gel? I stopped the electrophoresis right before the bromophenol blue dye ran off the gel, so would the protein be able to run faster than the dye?


It can, and it can also be within the line of the bromphenol blue, making it difficult to see

-syaniv-

which types of membrane have you tried? PVDF 0.2um is best for tiny proteins - and if you're not sure about loss, you might wish to find a standard with a smaller smallest protein, to be certain you're not running it off the gel. it's cheap insurance, and can save you lots of headaches smile.gif

I have used 16% tris-tricine for a 3.5kD protein; I don't think 15% will lose it completely...but I used to run the gels more slowly for small proteins. also, perhaps reduce your transfer time a bit; it could be that it's going through the membrane, even though your larger proteins (from the std) transfer OK

good luck

-aimikins-

Ok, thanks for the ideas guys, I will retry with a better marker, tris-tricine, and a PVDF membrane. I was using nitrocellulose before with a 1 hour transfer time at 100V. What type of transfer would you recommend, both for nitrocellulose and PVDF?

-Ra_-