western exposure one part of band darker - (May/03/2010 )

Hello,

Does anyone know why half of the band on western blot is darker than the other half when film is developed?
how much SDS buffer you should add to x cells?is there a ratio?

thx

Julie123 on May 3 2010, 11:37 AM said:

A picture for the first question might help. If you mean that bands on one half of a membrane appear darker than the bands on the other half, then there could be a number of answers: more protein in those bands is the most obvious answer (why- more protein loaded?, transfer issue? treatment causing up/down-regulation of gene?), other answers include loading issues, membrane not properly covered by antibodies/wash during probing steps, poor mixing of chemiluminescent substrate... etc.

I add 10,000 cells per ul of lysis buffer, I have done as much as 100,000 and as few as 5,000 per ul.

Julie123 on May 3 2010, 07:37 PM said:

is this a constant problem? may result from polutions of the developing machine or developing cassette

I see a similar problem and I'm not sure the cause. It seems like it could be from the transfer, but it's not always in the same place. Also it's only usually seen in highly expressed proteins.
See attached picture.

yoshimi79 on May 21 2010, 12:12 PM said:

Looks like an exposure issue to me, try laying down and lifting up your membrane a few times at the start of the incubation with the substrate, it should mix the water/buffer on the membrane with the substrate more evenly.

I generally mix the substrate around on the blot and then leave it for five minutes.

Do you have a preference for face up or face down in the substrate?

Looks like you are getting very close to a "burn-out" on many lanes. Reduce your primary Ab and see what happens.