Sample Buffer - (Jun/30/2008 )
After completing an IP, I accidently added 50ul of 6X sample buffer instead of my 2X sample buffer. Realizing my mistake, I added 10ul of 600mM DTT to give a final concetration of 10% SDS, 50% glycerol, 300mM Tris, 0.05% blue, and 100mM DTT.
The 2X buffer I usually use contains 8%SDS, 10% glycerol, 37.5mM Tris, a very small amount of blue and 5% BME.
Will the more concentrated sample buffer affect my IP? Would cause the proteins to not come off the beads? Or could it cause extra proteins to come of the beads?
I have already run these sample and I am finding bands interacting with my monoclonal FLAG blotting antibody that shouldn't be (the cells used for the IP were not transfected with FLAG). I am just wondering if this could be a result of my sample buffer.
The 2X buffer I usually use contains 8%SDS, 10% glycerol, 37.5mM Tris, a very small amount of blue and 5% BME.
Will the more concentrated sample buffer affect my IP? Would cause the proteins to not come off the beads? Or could it cause extra proteins to come of the beads?
I have already run these sample and I am finding bands interacting with my monoclonal FLAG blotting antibody that shouldn't be (the cells used for the IP were not transfected with FLAG). I am just wondering if this could be a result of my sample buffer.
with high concentration, your proteins will surely come off the beads. Extra proteins could be the protein G or A. or if you are using anti-flag antibody resin, then it will also come off. are you refering that you get the band in control or what. please write it clearly. if u get band in control, then it could be non-specific. you can do preclearing and more stringent washing.