Minimum volume antibody solution for Western blot in tray? - (Apr/09/2013 )
What's the least volume you can use for incubating a western blot with antibody in a tray? We have approximately 5 x 8 cm (so ~40cm^2) trays and I can get down to about 4-5mL before I start to notice problems (sometimes works, sometimes you get blank areas). Of course it might be problems due to something else but I assume it's because of the low volume. I used to use pouches when necessary. We don't have pouches and a heat sealer here but I think one of the other labs might. I'm curious what others have found in the minimum volume to use for incubation.
Yeah, 5 ml is about the minimum for that sort of area, probably too little in fact. In a pouch you can get down to 1-2 ml.
Here we use a tube roller and a minimum of 2 ml for a 40-50 cm2 membrane in a 50 ml falcon tube.
I'm with bob1, we use 50 ml conical tubes with 5 ml solution for primary antibodies.
Would a tray+rocker then work instead of a roller if we don't have one of those? It seems like it should give the same type of effect as a roller (solution continuously passing over). I had been using a horizontal shaker with a tray before. Maybe the horizontal shaker doesn't mix well enough with low volumes, but a rocker might.
Rocking or nutating trays work well.