Western Blot Mystery - Developed bands don't adhere to membrane

[cyrus]

I am trying to use commercial antibodies to recognize coral proteins. However I have run into some peculiar problems. After several attempts, I finally got my Ab to work and got a great membrane scan. However, since then I have not been able to get the developed bands to remain on the membrane. After allowing the bands to develop for 30 mins, I go to move the membrane to place it on a scanner. As soon as the membrane moves, all of the developed bands slide off, they are not adhering to the membrane! Even putting on new developer does not work, the bands will not come back after the initial development. I have tried troubleshooting the amount of non-fat milk used for blocking and during the Ab incubations, the Tween-20 concentration, new solutions, etc. to no avail.

The correct bands are appearing, they are just not adhering. Does anyone have experience with this or can shed some insight?

What I am currently doing is running a 12% bis/acrylamide gel, with a 4% stacking gel, with my samples and a rainbow marker, run 1hr 47min, at 120V. I then perform a semi-dry transfer at 12V for 45min, using PVDF membrane. I block overnight in PBS-Tween 20 0.5% (PBS-T) and 2% non-fat milk powder. The next day I put in my Ab 1:200 dilution, in PBS-T and 2% non-fat milk powder for 24hrs. On the last day, I do my secondary Ab for 1hr 1:2000 dilution in PBS-T, 2% non-fat milk powder, and normal donkey serum; followed by the tertiary Ab for 1hr in PBS-T alone at a 1:1500 dilution. I develop the membrane using 3,3',5,5'-Tetramethylbenzidine (TMB).
In between each step there are washings: 3 quick rinses with DI water, followed by 3 washes for 15min with PBS-T.

[admin]

Hey there Cyrus,
sounds like either an antibody issue, a membrane issue, a washing/buffer issue or a bit of all 3.

I would bet its a washing issue (#3). Have you tried using a better wash buffer instead of DI (distilled?) water - maybe TBST buffer or even PBS?

Also try to use less harsh washing/buffer conditions if you are and less rotation in the circular rotator - mine sometime gets pretty choppy and its not always a good thing.

[cyrus]

Hey Admin,

Thanks for the response.

For the washing, I do quick rinses with DI water followed by washes using PBS-T. Should I cut out the DI water rinses?

I have not tried just PBS without the Tween20, do you think that would make a difference? I did a test set last week, doing one membrane with PBS with no Tween 20 the whole way through and the membrane did not come out with anything at all.

As for the wash motion, I use a rocker that goes only back and forth, usually at a low setting, but I will try to turn it down some more as well.

[admin]

Yeah, try the experiment without the distilled water rinses. Do you use the distilled water tap dropping water on the blots directly? The distilled water (basically no salt) may disrupt the antibody-protein-membrane interaction. Thats my guess.

Let me know if that helps or what was the issue, very curious...

[cyrus]

Would making the solutions in distilled water or MilliQ water make a difference?