Western Blot - signal doesn't show up on film - (Sep/09/2004 )
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!
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
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 ?
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
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.
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
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.