Western bands fading out, pleeeese help - (Dec/09/2009 )
Hi all,
I have been doing western blot these days, till last week results were OK, now my bands are behaving strangely, 
ie, westernblot bands are fading out quickly befire capturing in a X ray film.we r using super signal west femto luminol enhancer solution (pierce) to develop the membrane.
Briefly we r running 70mg of sample in a 12% SDS-PAGE, after overnight transfer in nitrocellulose membrane (at 4oC), block with 5% nonfat milk in TBST for 1 hr and incubate overnight with primary antiboby (1:1000, cell signalling technology) at 4oC. after another 1 hr of secondary antibody (1:1000 cell signalling technology) treatement at room temparature, develop the membrane using the afore said substrate.
I request for your help 
, as any suggestions will help me in facing this problem
Thanking in advance
niveda on Dec 9 2009, 06:02 AM said:
I have been doing western blot these days, till last week results were OK, now my bands are behaving strangely,
ie, westernblot bands are fading out quickly befire capturing in a X ray film.we r using super signal west femto luminol enhancer solution (pierce) to develop the membrane.
Briefly we r running 70mg of sample in a 12% SDS-PAGE, after overnight transfer in nitrocellulose membrane (at 4oC), block with 5% nonfat milk in TBST for 1 hr and incubate overnight with primary antiboby (1:1000, cell signalling technology) at 4oC. after another 1 hr of secondary antibody (1:1000 cell signalling technology) treatement at room temparature, develop the membrane using the afore said substrate.
I request for your help
, as any suggestions will help me in facing this problemThanking in advance
Try diluting you secondary antibody more. I know for some secondaries using SuperSignal, it is recommended at least 1:30,000. If you have too much HRP present (i.e., too much secondary), you will use up the SuperSignal substrate very quickly. I use the pico kit, and there is a troubleshooting section in the manual that describes what else can be done if the signal burns out too fast.