running biotinylated sonicates with sds-page - (Jan/16/2007 )
I'm doing something that seems very simple-- but problems arise! If anyone can help, it would be great.
I am working with bacterial surface biotinylation and trying to get my negative control (biotinylated sonicated lysate). When I do the biotinylation with sonicated and whole cells and then run on the gel, I'm losing my sonicated load-- see very few bands with the sonicated sample (after amido black stain) as compared with the surface biotinylation lysate sample. Are they not entering the gel?? Is this just a general gel loading problem?
The biotinylation was stopped with tris-HCl and then dried down, resuspended in gel loading buffer.
no biotinylated protein capture on streptavidine agarose before to load on the gel?
I don't understand why your biotinylated extract doesn't migrate. It should. I never had problems with that, unless when I wanted to capture the biotinylated proteins first.
I don't understand why your biotinylated extract doesn't migrate. It should. I never had problems with that, unless when I wanted to capture the biotinylated proteins first.
No capture first, just wanted to run the biotinylated sonicated lysates on the gel, then blot w/ SA-HRP after to show that my sonicated fraction shows many more bands than the unsonicated frac-- that the biotin linker is somewhat specific for surface only. Should be able to run both fractions, and get a sample of all the proteins in the bacterial cell. However, before blotting and after transferring, I put on amido black and noticed almost no bands in the sonicated frac, so I haven't moved on with the blot. Obviously something went wrong. But I have a hard time believing that it has something to do with the biotin specifically, so that's why I wonder if it's just a more general problem of drying down the tris and having too much salt in the sample or losing a lot of my load. But I don't get it. Will try the same thing with unbiotinylated cells and see if it works better.
I don't understand why your biotinylated extract doesn't migrate. It should. I never had problems with that, unless when I wanted to capture the biotinylated proteins first.
No capture first, just wanted to run the biotinylated sonicated lysates on the gel, then blot w/ SA-HRP after to show that my sonicated fraction shows many more bands than the unsonicated frac-- that the biotin linker is somewhat specific for surface only. Should be able to run both fractions, and get a sample of all the proteins in the bacterial cell. However, before blotting and after transferring, I put on amido black and noticed almost no bands in the sonicated frac, so I haven't moved on with the blot. Obviously something went wrong. But I have a hard time believing that it has something to do with the biotin specifically, so that's why I wonder if it's just a more general problem of drying down the tris and having too much salt in the sample or losing a lot of my load. But I don't get it. Will try the same thing with unbiotinylated cells and see if it works better.
what is working :
take 5.10^9 bacteria lysed in 200µL PBS + 1% triton X-100. centrifuge, add biotin 2 mM to the supernatant, and then add tris 50 mM (final concentration).
load 10 -30 µL on a gel and perform western-blot.