no black band but empty stamp of the protein - instead of black bands in xray film, the space is empty with a stamp o (Apr/24/2010 )
Hi all,
Everytime, when my marker got stamped in the xray film, i was more than happy 
because it becomes easy to examine the size. But this time, instead of black bands, i have similar stamps of protein in protein lanes 
. Is there any way i can solve this? the band stamp is exactly where itīs supposed to be and the western has run perfectly. I donīt understand why itīs a transparent shadow?
many thanks in advance to suggestions...
kedar
Whenever this has happened in my lab, it is because it is OVERexposed. Try probing with less antibody, using a diluted developing fluid, and/or exposing for less time.
kedar on Apr 24 2010, 08:56 AM said:
Everytime, when my marker got stamped in the xray film, i was more than happy
because it becomes easy to examine the size. But this time, instead of black bands, i have similar stamps of protein in protein lanes . Is there any way i can solve this? the band stamp is exactly where itīs supposed to be and the western has run perfectly. I donīt understand why itīs a transparent shadow? many thanks in advance to suggestions...
kedar
I assume you are putting on the ECL reagent at your bench and walking down to the darkroom. I've shown two people in my lab this...your signal is so strong that you are burning through all the reagent by the time you reach the darkroom. Do yourself a favor and mix up a bit of the ECL reagent in an eppy-tube. Take your membrane, the ECL, a bit of saran wrap or transparency film and a pair of forceps to the darkroom. In the darkroom put the ECL reagent on the membrane and turn off the lights. I'm willing to bet you will actually be able to see the blue light coming off the membrane.
rkay447 on Apr 27 2010, 07:51 PM said:
kedar on Apr 24 2010, 08:56 AM said:
Everytime, when my marker got stamped in the xray film, i was more than happy
because it becomes easy to examine the size. But this time, instead of black bands, i have similar stamps of protein in protein lanes . Is there any way i can solve this? the band stamp is exactly where itīs supposed to be and the western has run perfectly. I donīt understand why itīs a transparent shadow? many thanks in advance to suggestions...
kedar
I assume you are putting on the ECL reagent at your bench and walking down to the darkroom. I've shown two people in my lab this...your signal is so strong that you are burning through all the reagent by the time you reach the darkroom. Do yourself a favor and mix up a bit of the ECL reagent in an eppy-tube. Take your membrane, the ECL, a bit of saran wrap or transparency film and a pair of forceps to the darkroom. In the darkroom put the ECL reagent on the membrane and turn off the lights. I'm willing to bet you will actually be able to see the blue light coming off the membrane.
I have just the same problem with actin bands. And the problem persists even if I expose the film for a few seconds. The dilution of actin antibody i use is 1:2500. Should I dilute it more?
I would appreciate it very much if anyone of you could help me solve this problem. It's very important for me to have good actin bands as I use them to calibrate the density of the other protein bands in case of incorrect loading.
Thank you very much.
Olga
I have just the same problem with actin bands. And the problem persists even if I expose the film for a few seconds. The dilution of actin antibody i use is 1:2500. Should I dilute it more?
I would appreciate it very much if anyone of you could help me solve this problem. It's very important for me to have good actin bands as I use them to calibrate the density of the other protein bands in case of incorrect loading.
Thank you very much.
Olga