Troubleshoot: Western; no bands appear in reduced lanes - (Jan/21/2007 )
Hello,
This is my first post here, though the site has always been one to come to if ever in case of dilemma.
Well, I am in a dilemma, so was wondering if anyone could assist.
After having some issues with my Westerns, I went back to square one. That means remaking all the buffers and solutions.
I prepared some lysates from three separate cell lines expressing my protein of interest, and ran them in the following manner:
1. Lysate (50ug) non-reduced
2. Lysate (50ug) b-merc (5%) reduced
3. Lysate (50ug) DTT (20mM) reduced
I repeated this loading for each of the cell line lysates. However, on exposing my gel, only bands appeared for the non-reduced samples. This is simply odd, because standard lab protocol is to reduced the samples using 5% beta-mercaptoethanol for this particular protein. And it has been working for years.
Does anyone have an idea why my reduced samples do not appear?
Thanks,
William John.
Just wondering...
Did you boil your reducing samples?
Did you boil your reducing samples?
The reduced samples were treated to a 90 degrees C heat block for 5 minutes. I also Ponceau'ed, and the lanes were evenly loaded.
William.
Hi..,
can you detect any other protein in your reduced samples, let's say tubulin or GAPDH or anything else?
Maybe one of your colleagues is willing to perform the WB for you to see if you make a mistake of which you are not even thinking currently.
This can actually be very helpful before going crazy.
Good luck
is your antibody designed for western-blot (i.e. for denatured antigens) or for ELISA, IC ?
can you detect any other protein in your reduced samples, let's say tubulin or GAPDH or anything else?
Maybe one of your colleagues is willing to perform the WB for you to see if you make a mistake of which you are not even thinking currently.
This can actually be very helpful before going crazy.
Good luck
I haven't probed for house proteins, but did probe a duplicate membrane with antibody directed against glutathione-bound proteins, and got the expected pattern (many bands). I also did a Ponceau, and the loadings were even and there was nice banding there as expected.
Will have to see if someone can add some samples to empty wells at some point, but right now I am doing a Western with boiled and non-boiled samples to see if that is causing the adverse effect.
William John.
The antibody is for Western blotting. I've also used it recently for immunofluorescence, and the bands that are coming up on the membrane are at the correct weight.
William John.
I'll be exposing my latest membrane today with boiled and non-boiled samples, but any ideas would be appreciated,
all the best,
William John.
well, mystery seems solved for now... my title for this post should now be amended to 'no bands appear in boiled sample lanes', as the non-reduced sample came up with a nice band, however non-reduced + 5mins at 90 degrees C didn't. Same with reduced samples.
how anomalous, considering this protein has come up for years using the protocol of boiling the samples. something must have changed, but i'll suffice in being happy if my band comes up without extra necessity.
william john.