Strange run or transfer of proteins at 15% PAGE (not band but spot) - (Jan/28/2008 )
I have a problem with run or transfer of proteins at 15% SDS PAGE and 15% native PAGE. In both cases (SDS or native PAGE) there is the same problem - I do everything (run, transfer, primary, secondary antibody incubation etc.) in the same way as at a 8, 10, 12% PAGE, but after the visualization (developing) with ECL I see instead of (black) bands (like at 8, 10, 12% PAGE) rather circles or circle spots (signal is good, clear, strong but there is no band but circle spot). When I stain the gel with coomassie blue there are quite normal bands.
What can be problem? Do you see normal bands at 15% gel after PVDF ECL visualization?
Thanks for advice,
I have never heard of this. Can you perhaps show a picture? My 15% gels work the same way like 10 or 12,5%, everything looks the same. How big is your protein? It does not get stuck up in the wells, or? Can you detect a loading control protein like GAPDH or Actin as a band?
U should stain ur membrane with ponceu red after transfer in order to verify the proteins binding on the membrane.
then u can distain the membrane and move on to western.