bubbles and overheating of western blot - (Nov/10/2008 )
For the past month or two, every time we perform a Western blot it overheats and bubbles while running. If we try to transfer to gels at one time the one farthest from the ice block has splotches all over it. (I make sure to get all of the air bubbles out.) We have changed the buffer several times and it does not seem to help. We run it at a constant voltage of 100 V for one hour and I noticed that the amps reach about 0.87 A. I don't know if something is wrong with the power supply. We also have troublke running gels and it used to over heat. We are also having a problem with trying to detect albumin in our samples. The bands are fuzzy and unclear when we develop the blot after treating with a primary antibody for albumin. Any suggestions would be thoroughly appreciated.
may be something wrong with the buffers?
may make some fresh buffers.
Have you tried to reduced your voltage and let the gels run longer (the same for the transfer)? Maybe this will give better results, even if it takes a bit longer. And run your gels in the cold room.