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.