"Sandy WB" - (Oct/18/2010 )

I have been spending my life recently trying to make my WBs work again...
They look "grany"...not sure how to explain but basically, very dirty, full of non specific bands and with a "sandy" texture...

I am trying to detect pAkt (and also pS6K) and I cannot get a decent signal.
I've been following a protocol given to me by my boss, and apparently it seemed to work for him.
I prepared all my solutions (Tris buffers for the gel, Transfer buffer...) from scratch more than once but no result.
I used a different secondary to see whether the problem could come from there, but the result was the same.
I blocked in 5%milk-PBST or 5%BSA-PBST, and again nothing.
I tried to cast my gel fresh on the same day I am running them (and not the day beofre, storing at 4C as I was doing before), nothing...
I am really desperate to get these WB to work because they're really crucial for my PhD thesis and I don't have that much time.

I am thinking about other possible options:
-using TBS rather than PBS
-changing the membrane (borrowing some other nitrocellulose membrane, mine could have gone bad misteriously)

Can anyone please give me any suggestion?

Thanks a lot

-Fly78-

How are you doing the transfer? I've seen things similar to what you describe when using the X-Cell blot module (from Invitrogen) and you forget to use Whatman paper in your transfer stack such that the gel and the membrane are in direct contact with the sponges -- this results in a "pebbly" or perhaps "sandy" look...

-HomeBrew-

HomeBrew on Mon Oct 18 19:56:26 2010 said:

I am using the Trans-Blot Semi-dry (BIORAD) and I use three layers of filter paper (pre-wet with transfer buffer)on each side...

-Fly78-

just a suggestion: clean your sponges carefully. With mild acid or sth like that. After longe usage they can be soiled by proteins.

-ksturm-

I am not actually using sponges, as I said, I am doing a semi-dry transfer and use 3 layers of filter paper on each side...

-Fly78-

Sorry, my fault. Biorad semidry is actually very good system. 'sandy' appearance dues to problems during transfer. Beside nitrocellulose pvdf-membranes are recommended for the biorad System. But 10-15% of protein pass pvdf-membrane without binding. What is your transfer Buffer? Unspecific bands are due to blocking or primary ab. Some people add Tween20 to their blocking solution.

-ksturm-

I've had this problem before when trying to blot for a membrane protein. I blocked membrane in 5% milk overnight to get rid of the grainy appearance on film. Hope this helps.

-elaila-

What developing system are you using (HRP, alkaline phosphatase?), what sort of film?, how old are the film processor chemicals?

-bob1-

Fly78 on Mon Oct 18 17:10:35 2010 said:

In the past I've run gels where the glass plate over the front has come loose. Sometimes this causes protein to leak out of the front of the gel and it can make the blot look a bit grainy.

Also, to maximise your blocking, try diluting antibodies in blocking buffer rather than just PBS - should help to minimise background. And (you probably do this already) make sure you wash in PBS with Tween in it and wash at least three times with high volumes of wash buffer.

As for non-specific bands, are you lysing your cells in buffer with a protease inhibitor? Sometimes breakdown can occur during lysis. I've also noticed I get some non-specific bands around the same weight as my protein of interest if the cells are too highly confluent when I lyse them.

-steffi333-

Fly78 on Tue Oct 19 14:08:28 2010 said:

I am not actually using sponges, as I said, I am doing a semi-dry transfer and use 3 layers of filter paper on each side...

Try using 6layers of filter paper on each side. So 12 layers per gel.

-Say Chi Sin Lo-