Bands on Western are not sharply defined - (Feb/02/2005 )

Hi guys, hopefully someone would have an idea about it:
many times after running Western and exposing the film, the bands on the film appear "out of focus". My colleague is able to get the bands a lot more difined, sharp so to speak. Do you know what I might be doing wrong? I tried to cut down the time between SDS PAGE and transfer to a minimum, but that made no difference. What else should I try?

If the film moves in the film cassette during the exposure, then youll get smeared bands. This is especially true for very very short exposures. It took a few times before I was able to do a 1 second exposure with good results.

Also, I make sure I lay my film down flat in the cassette, then bring the blot to the film via the hinged lid. When I started doing westerns, I was attaching the blot to the back of the cassette, then lying the film onto it, then closing the cassette. The former ensures the film doesnt move
Additionally, and unrelated to the exposure of the film:

Ive heard some people adding glycerol to the acrylamide gel recipe, and supposedly it makes sharper bands. Also Ive heard that some people run the stacking gel at a very low voltage, compared to the normal voltage one might use in the running gel.

Hope this helps you out, and someone please correct me if Im wrong about these tips - I do not employ these practices when running gels.

Maybe you should try loading the samples very slowly and cautiously into the wells, allowing the sample to concentrate only at the bottom of the well. Also make sure that none of the sample escapes as you pull your pippete out.

Hope this helps!

hey snolan6
that was a really useful tip about the film in the cassette; I shall try that out soon.

could anyone help with a slightly unrelated problem: i get dreadful streaming on the gel (and western, therefore) with SDS lysates, which are absent on CHAPS and MPER lysates, all of breast cancer cell lines. would be grateful to know what protocol seems to give good clean lysate with SDS lysis buffer (re, specific steps such as sonication or heating etc).

cheers!
sean.

That's the problem with SDS lysates, the SDS reacts with the DNA
Just sonicate your samples for 1-2 s... or add DNase to your sample buffer..

good luck..

... and

You can get good bands in SDS-PAGE, if you cast the stacking gel fresh and make sure your buffers have the right pH....

thanks spraq. think I noticed some improvement in the lysate after I tried the sonication. yet to see the western.

sean.