SDS-PAGE trouble with protein concentration - (Sep/12/2012 )

Hey Everybody,

Here's the set-up: I'm doing some SDS-PAGE on some samples, obviously. I am using a TCA method where I obtain a pellet and resuspend in Laemmli's/2BME and boil it prior to loading on the gel. Prior to this, I have been trying to normalize the amount of protein to an added protein of known quantity in each sample, but it's not going so well. I am running into problems with protein concentrations.

So, I'm thinking of doing a Bradford but am confused on it a little. When would I carry out the bradford? I don't stain with coomassie blue and since it's in pellet form prior to sample buffer addition, I don't want to mix the bradford reagents with the pellet.

I'm pretty new to this stuff and have been trying to figure this method out pretty much by myself. So, go easy on me. I'm looking for some protocol on how to do the bradford or equivalent protein concentration technique with a prepared pellet prior to running the sample through SDS-PAGE.

Thanks and let'see what you got.

you can prepare laemmli sample buffer without bromphenol blue, resuspend the pellet and boil (as before), take a sample for the bradford and add bromphenol blue to the remaining sample and run.

after you crash out the protein with your tca method you can resolubilize it with RIPA buffer and run a BCA assay on it. or if you know you're only after the cytosolic proteins you can use tris hcl buffer to resuspend and do a bradford.

if you run bradford on samples in laemmli sample buffer you better make sure to dilute it a lot because bradford only tolerates up to 0.125% sds (it interferes with protein surface interactions... to say the least).