hi, i have been doing western blots for about a year now. i used to get a great gapdh signal, but now there is no signal at all and if there is, it will be very weak. the protocol is the same i only changes the % of the acrylamide gel from 10% to 15%.I boil the proteins with loading buffer at 100 degrees for 10 min, I load in each well 20 ug of proteins, and run at 100 v. the separation of the protein marker is very clear. the transfer is also good as well at 80v for 90 min. i block the membrane in 3% BSA and tween for 2 and half hrs. primary Ab concentration is 1/3000 and that of the secondary is 1/10000. could anyone help me to find out the problem ?!