Hello,
I have been trying to detect very low molecular weight proteins (14-17 kDa). I used a 15% polyacrylamide gel and I couldn't see my protein. With a pre-cast gel (4-12% gradient polyacrylamide gel) and a different chamber, everything went fine and after the transfer I have detected the protein of interest. As a consequence, to better understand the cause of this problem I have re made both the separation and stacking buffers and verified that they have the correct pH, APS 10%, used a new bottle of TEMED and the running buffer (which also has a correct pH). I also tried to make a gradient polyacrylamide gel (8-16%). What can be the problem? Can the low mA (we use constant voltage) be the cause of this problem?
Hi Marta Doglio
How much are you running the 15% SDS PAGE, for visualizing your protein of interest?
I am myself working on a protein of m.w. ~18 kDa, and for 15% PAGE, I stop the run as soon as the tracking dye is still at a 0.3-0.5 cm distance from the edge. I am able to visualize a neat protein band.
Do you have any ladder? It would really help to actually see how samples move across the gel. It seems like the problem is with your gel as everything else is same for the pre-cast gels. Check the gel making protocol if you missed anything. Hope that helps!
Taufeeque Ali Thank you for your answer! I am able to distinguish all of the bands of the marker very well, but with blue comassie staining I can't see the low molecular weight proteins.
Aakriti Singh thank you for your answer! I let the tracking dye exit the gel (the run goes on for about 2.30 h). At this point the 10 kDa band of the marker (the last one) is at 0.5 cm from the bottom of the gel.
Marta Doglio, My Suggestion would be don't let the tracking dye exit the gel (Also, the run time seems too long, what voltage?
Marta Doglio That again narrowed it down to the Gel you made. I would suggest run another experiment side by side, with pre-cast gel and the casted gel and use the same sample to load in both (Prepare loading sample together in one vial, not separate).
it is not usefull not to let the tracking dye exit the gel; there is no (resolved) material in front of the blue dye. you can run a 20% acrylamide gel . For small molecular weight protein you should run a tris-tricine gel to have a nice resolution (maybe the precast gel you use was a tricine gel) Also small protein are not stained as well as larger one so maybe you do not have enough of the protein you are looking for to see it by coomassy staining. Did you try western blot?
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