5 replies to this topic

[kedar]

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 it´s supposed to be and the western has run perfectly. I don´t understand why it´s a transparent shadow?

many thanks in advance to suggestions...
kedar

[epagano]

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.

[rkay447]

kedar, on Apr 24 2010, 08:56 AM, said:

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 it´s supposed to be and the western has run perfectly. I don´t understand why it´s 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.

[olga_m]

rkay447, on Apr 27 2010, 07:51 PM, said:

kedar, on Apr 24 2010, 08:56 AM, said:

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 it´s supposed to be and the western has run perfectly. I don´t understand why it´s 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

[fysio lab]

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
[/quote]

Dilute primary or secondary more...your signal is too strong

[Another Jake]

Two words:
ECL Plus.


... I guess that's actually one acronym and one word, but psh.


Loading less protein can help too if you still have issues after diluting your primary.  If it helps, I dilute some of my primaries to 1:7500 or even in one case, 1:15000.

Edited by Another Jake, 05 May 2010 - 09:01 PM.