Resolving LMW proteins

Malcolm Moos Jr., M.D., Ph.D. moos at helix.nih.gov
Fri Apr 1 18:29:07 EST 1994


In article <29MAR199420201323 at bioch.tamu.edu>
erich at bioch.tamu.edu (Eric Hebert) writes:

> >> Hi there,
> >> 
> >> I'm trying to run a 8 kd protein on a SDS get but I'm having some problems
> >> getting it to show up due to its small size.  I've tried 10, 12 and 15
> >> percent gels but the but the protein doesn't seem to be there ( the problem is
> >> obscured by the fact this protein is being made after an induction and the coli 
> >> proteins interfere with seeing the bottom of the lane where the 8 kd protein
> >> would presumably run.)  I've tried running the gels without loading dye in the
> >> sample buffer thinking that the dye front would obscure the 8 kD band.  This
> >> didn't help.  If anyone has any suggestions about resolving low molecular
> >> weight proteins please respond by posting in this group or emailing me
> >> directly.  Thanks.
> >> 
> >> Arul
> >> ajayaram at pearl.tufts.edu

Arul,

Some further thoughts on analysis of low Mr proteins. First, I confirm
that the Tricine system will work well and reliably for proteins in
that size range. A straight 10%  or 10-20% gel should do nicely. The
gels of this type available from Novex perform very well. (No
commercial endorsement is implied and this view is that of the author
and not of the Federal Government or any of its agencies &c.) The
staining procedure in an earlier posting will work well; the issue is
diffusion of low Mr peptides from the gel before fixation. Another
alternative, in case you don't have sulfosalicylic acid on the shelf,
is to place your gel in 40% ethanol, 10% acetic acid, 0.1% Coomassie
Blue R-250 and immediately microwave it until hot but not boiling. No
open flames or sparks, please. Shake at room temp 15 min and transfer
to 10% ethanol, 7% acetic acid. Microwave as before, shake another 15
min, and repeat. I have stained 2 KDa peptides this way.



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