I don't think your problem is the size of the protein. We have been
doing western blots on a 8K protein for many years using standard Laemmli
gels and standard semi dry blotting techniques. Our protein has a pK of
around 9.0.
If the protein resolves on your gel and is positive with the
antibody on direct dot blots to PVDF, then your problem is probably in the
transfer. The usual fix for this is to adjust methanol concentration and
time. Methanol strips the SDS from the protein but some SDS must be
retained for transfer to occur.
>Hi,
>I would like to know if anyone has experience with electroblotting, and
>western detection of very small proteins (2-8 kDa). I am running 16.5%T,
>6%C tris-tricine gels with a 10%T spacer gel. These give reasonable
>resolution. I am having trouble getting reproducible transfer of these
>proteins (standard tris/glycine/20% methanol transfer buffer) and am
>getting no signal on western blots. A control protein of 10 kD gives a
>positive on the western with the same antibody (and the small proteins
>contain the epitope to which that antibody was raised). I have tried small
>pore (0.1 um) nitrocellulose and PVDF membranes. I would appreciate any
>suggestions.
>
>Thank you, Susan
>
>Dr Susan Rowland
>Department of Microbiology
>University of Connecticut Health Center
>Farmington, CT, 06030, USA
>
>Email: rowland@panda.uchc.edu
>FAX: 1-860-679-1239
>PH: 1-860-679-2203
Gregory A. Grant
ggrant@pharmdec.wustl.edu