Film Black Western Blot Glowing Dark

[gandalfthegrey]

Hello,

my blot was glowing in the dark when exposing and my film came out black from the machine.

What should I do to fix this?

cheers

[Towe]

Hi Gandalf
Maybe you can use your magic staff and put a nice spell on your membrane. Or more seriously, try to wait fore a while before going to develope the film. If you use ECL you might wait for 30 minutes or 1 hour and then try to develope again. You might have used to much secondary antibody.

[gandalfthegrey]

LOL... thanks for the information. I think I may have added too much secondary antibody.

WoW.. do you play that game a lot (in the lab maybe jk ) ?


thanks that tip is a good one

[Towe]

I used to play it a lot but since I got a little daughter, my time for leveling is now only a couple of hours a week and I haven't installed it at my working computer. Jet.

[bwbrian]

A little too much secondary? If your blot is visibly glowing, you need to dilute your secondary about 100-fold. As a bonus, with more dilute secondary, you'll get better specificity and much more stable signal - which equals more reproducible results. Your exposure to film should take 30 seconds-5 minutes. If it's significantly less than this, you are hurting the reproducibility.

[ofir]

Hi
I can suggest 4 different ways that you can try in order to get a better result:
1. Dilute your secondary antibody
2. Put the ECL for less time (let's say 60 sec)
3. Dilute your primary antibody
4. Try using two films together
By the way do anyone have any suggestion on which software to use in order to quantified bands ?