Western blot bands fade out - (Jun/19/2006 )

Hi all,

I am doing western blotting and getting good bands but after 24 hours the bands fade out. Even the marker bands fade out. I am using Trans-blot NC memberane from Bio-rad. I am unable to figure out the reason. Can anyone help please.

Thanks

what are you using to visualize the bands? is it light sensitive? does it rely on a reaction? with substrate that can be used up?

more details are necessary.

Hi,

My secondary antibody is alkaline phosphate and I am using napthol phosphate substrate. Even if my protein bands fade out atleast the marker band should be stable. But my marker bands also fade out.

Thanks

If you are talking about the bands fading out on Film using a film processor...

then I am pretty sure there is a problem with the chemical mix used in your processor. Get it cleaned and add fresh developer fluid.

It is not a problem with your protocol.

Hi,

I am developing the blot on the NC membrane itself. Its like the standard old old way of doing blotting after transfering you develop the blot by adding your primary, secondary and finally enzyme conjugate and visualize your bands on the membrane itself.

I am not using the film processor. I just dont expect my markers band to fade out. The bands again become visible if we wet the mebrane with water.

the naphthol phosphate apparently bleaches over a relatively short period of time. why don't you try bcip + nbt as a color depositing substrate for the alkaline phosphatase conjugate.

Thanks, I wll try to use bcip/NBT and see if the problem resolves.

I use NBT/BCIP with alkaline phosphatase too.
But I think this will also fade with time.
Why don't you just scan the membrane after developing to get a lasting copy of the result...

Scan the blot and then it does not matter that the blot faded or got lost, or whatever.