Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Western Blot - signal doesn't show up on film


  • Please log in to reply
6 replies to this topic

#1 CRDD

CRDD

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 08 September 2004 - 11:41 PM

Hi!
i encounter problem everytime when i expose the bands on x-ray film from nitrocellulose membrane. i don't see any images on the film but the bands are there on the membrane ( which i can see in the dark). so i guess the problem comes from the x-ray film or the solution for film development.
i'm currently using Invitrogen product for blotting and kodak MXB film+ kodak GBX developer and replenisher. i'm not sure whether these two products are incompatible or something else??
do u hav any ideas bout this? kindly reply this message. your help is very much appreciated. Thank you!

#2 jadefalcon

jadefalcon

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 223 posts
1
Neutral

Posted 09 September 2004 - 03:16 AM

hi

bands you can see with the naked eye have to give you definitely a signal with your film. We worked with amersham hyperfilm or kodak films, and they work more or less all the same.

So maybe your films or your developing is amiss. try to take one film to the light outside your darkroom and the delvelop it. if it's not totally black, you know where to start.

mike
--- He who finds typos may keep them! ---

#3 CRDD

CRDD

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 10 September 2004 - 02:21 AM

hi

bands you can see with the naked eye have to give you definitely a signal with your film. We worked with amersham hyperfilm or kodak films, and they work more or less all the same.

So maybe your films or your developing is amiss. try to take one film to the light  outside your darkroom and the delvelop it. if it's not totally black, you know where to start.

mike

That was an interesting idea. I'll give it a try. but what is the theory behind it ?

#4 jadefalcon

jadefalcon

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 223 posts
1
Neutral

Posted 10 September 2004 - 02:41 AM

the theory behind??

rays(x-ray) or light (sun, lamp, chemoluminescence) will blacken your film (like photography). The more rays/light, the darker the film. So if you don't get anything with your (sun) exposed films, you know either your films or your developer is bad. try to renew your developer and try with another exposed film, if not black, it the films...

mike

Edited by jadefalcon, 10 September 2004 - 02:41 AM.

--- He who finds typos may keep them! ---

#5 BryanW

BryanW

    member

  • Active Members
  • Pip
  • 17 posts
0
Neutral

Posted 10 September 2004 - 11:14 PM

If you can not see the band on the film after 3-min exposure, either the antibody you used is not good or there is no antigen in your sample.
HRP principle is all the same and very sensitive.

#6 jadefalcon

jadefalcon

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 223 posts
1
Neutral

Posted 11 September 2004 - 05:25 AM

But CRDD said he sees the band on the blot directly, but then there's no signal on the film - so I would say the fault is with the film/developer, not with the blot (antigen/antibody/HRP).

mike
--- He who finds typos may keep them! ---

#7 CRDD

CRDD

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 13 September 2004 - 07:05 PM

To Mike, i get what u meant by exposing to light. i agree to ur idea.
To Bryan, i think it wasn't the problem of the antibody because i could see bands on the membrane. but still, thanks for ur reply as u were trying to help out.

maybe i really have to change the film.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.