Western Blot troubleshooting - (Jun/03/2005 )

Hi I need help
Ok this is it...
When I develop my westerns (using chemi) at the place where my bands are supposed to be I just get a nice black outline of the band. It is white inside this outline. Essentially instead of getting a black band I am getting a white band

any ideas??

you mean you have a "positive" image of your membrane???

Hey,

I had this problem, too.

I think you might have overexposed your membrane to the film (the film literally "burned away" at these points).
You could try to load less protein, use a higher conentration of secondary or primary antibody or to expose the film shorter to the membrane.
Good luck.
Cheers

äähhhh....

what I meant was: you could use a higher dilution of your antibodies and not a higher concentration...

Yeah a positive image.....

I exposed it for just a couple of seconds ...infact did not even close the cassette. Also when I leave the chemi to quench and then expose it to the film then all i get is the outline which gets lighter and lighter to the point that I get nothin' at all on my film.. no outline none whatsoever ..

I think the higher dilution of the antibodies make sense...lemme give it a shot. thanks a lot for your input guys..
have a good weekend for the lucky ones who r not working

p...

Hi I need help
Ok this is it...
When I develop my westerns (using chemi) at the place where my bands are supposed to be I just get a nice black outline of the band. It is white inside this outline. Essentially instead of getting a black band I am getting a white band

any ideas??

reduce the amount of secondary antibody...

I have experienced the same when I had too much protein/antibody like with loading controls (actin) I think you just have to load less extract and/or reduce the amount of the primary antibody.

Regards,
Antonio

Hi I need help
Ok this is it...
When I develop my westerns (using chemi) at the place where my bands are supposed to be I just get a nice black outline of the band. It is white inside this outline. Essentially instead of getting a black band I am getting a white band

any ideas??