I have been trying to identify analogues to human growth factors in biological secretions. I am having trouble detecting EGF, even in the positive control. I have tried increasing the gel to a 20% gel, decreasing the transfer time to 45 minutes and using 2 PVDF membranes to try and catch the EGF if it manages to transfer through the first sheet. We are using reducing western blots and running at 60V through the resolving gel and 40V through the stacking gel until the loading front is about 2/3 the way down the gel. It may be the case that our positive control is simply not working, but before I try and repeat it, I would like to know if there are any modifications that should be made to the western blot method when looking for small 6 kDa proteins?
I have used standard 15% SDS-PAGE (reducing conditions), run until the dye front reaches approx. 1 mm from the edge of the gel, and WB with nitrocellulose under standard conditions (per BioRad's Trans-blot module instructions, used with their mini-Protean cell), and have had no problems detecting human EGF. The difference is that this is a recombinant protein, and the amount loaded is enough for visualization using Coomassie staining alone. Perhaps your problem is an issue of sensitivity? I would expect that you'd need to detect much lower quantities...
I use Tricine instead of glycine in SDSPAGE for small proteins. And also use the smallest pore size for your membrane.
http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.4.html
Small peptides retention on blot could be improved cross-linking by FA or GA just after blotting. It might be help, if did not disturb the detection..
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