I'm trying to figure out how to not need so much antibody for our Western blots. Right now, we use 10 mL of buffer to dilute our antibodies in. Any less than that, and the membrane isn't submerged in the solution. Any way to use less buffer/antibody?
Camille, if you have a recipient where the membrane fit exactly, you can use 2-3 mL of diluted Ab. We routinely put membranes in a parafilm fragment bent at the half, seal two borders with a heat sealer, add the Ab and seal the parafilm. We use this small volume for expensive antibodies or antibodies to be used diluted 1/1000.
Hi Trautman!
Slice your membrane using ladder as guide. This will result in smaller size of membrane then use smaller size containers for further steps.
I'm not sure whether you're already doing this or not, but this is how I get my membranes properly soaked in around 3-5mL of antibody.
Hope it helps.
Good luck!
Hi Camille,
Maybe you are already doing this, but you can incubate your membrane (or membrane slice) with the antibody overnight instead of 1-2 hours. Then you can reduce the concentration of your antibody.
It's also possible to freeze and reuse your antibody diluted in the buffer. I use blotto A to dilute my antibodies and I can reuse it up to 3-4 times with a good efficiency.
You can reduce the volume of the incubation solution to 5 mL, this will reduce the amount of ABs needed by 1/2.
- Badges
- Science method