white band on WB - Western blot (Dec/01/2005 )

Hi Guys

this may sounds stupid but i'm new to western

My film instead of being white with blck bands is gray with white band (my marker and the putative band i'm looking for)....(look like the opposite)

I'm using a std protocol...wash/block 30 minuted PBS +tween (.01%)/priamry (1:1000) one hour/3 washes PBS tween/ secondary (1:5000)/wash develpment with ECL from PIERCE.

The film is a medical film x ray from Fujii....(super RX)

Any obvious mistake???



Luca,


Thanks

I saw something on here fairly recently...I would do a quick search of this sight for "ghost band" and I think you will see a couple threads (although off the top of my head I'm thinking it's wayyyyyy too much signal, but I'm not sure)

good luck!

Greetings!

I have seen these bands a few times, too. According to my handy Pierce Western Blotting handbook and troubleshooting guide , a possible cause is that the antibody concentrations are too high, especially the HRP conjugate.

" appearance of white bands are indications that there is too much HRP in the system"

Good luck!

monkeybaji

Too much protein concentration loaded.Try diluting

I have to think the reason would be similar. although it's different chemistry, still they all work in essentially the same fashion and the problem would be too much reagent/component leading to rapid saturation of the signal, no?

Does it really block : 0.01% tween?
I block with 1-5 % of BSA, non fat dry milk, fish gelatine... proteins.
I use tween to help to wash, as a detergent.

Could it be the reason that you have all your signal on the membrane, between your proteins, making this negative signal?