ABout the solution in Western blot and SDS? - (Sep/12/2006 )
There are few questions which i do not understand:
1. Why some journals pointed out that milk powder for blocking is suitable for monoclonal anitbodies but BSA is suitable is suitable for polyclonal antibodies?
2. WHy milk powder has blocking effect?
3. For the solution "TEMED", the pale yellow color is shown, can it still be used?
4. For making the dye in SDS, after i added the bromophenol blue powder, initially it turn blue, after i vortex the solution, it became dark green in color (terrible), Would anyone can tell me how to make a good dye for running the SDS sample?
Sorry for asking so many questions
I'm sure there is more technical answers out there but my understanding is as follows:
1/ I don't believe there is a difference between blocking between monoclonal and polyclonal antibodies ( I tend to use milk for both). However, there are some circumstances that I find that using BSA is better - primarily when using phospho-antibodies (milk contains phosphatases) and I sometimes find BSA gives less non-specific background.
2/ Milk is a protein rich solution and contains a number of proteins including caesin. These proteins can sit on proteins that are 'sticky' and mask these non-specific binding sites from the antibody.
3/ I'm not sure what you mean
4/ I assume that you are making a 2x or 4x concentrate dye. Under high concentrations bromophenol blue appears dark green or even red. I'm sure your dye is OK and when added to samples returns to a 'normal' blue colour. If you are worried about it you can drop the concentration of bromophenol blue.
Hope this helps
3) TEMED appears yellow sometimes when it becomes old. You can still use it but it will take longer for your gel to polymerize
to expand on scott's response:
1) milk contains phosphoproteins (casein is highly phosphorylated)
2) milk and bsa and other blocking agents will block unoccupied sites on the membrane to prevent antibodies from binding directly to the membrane
4) your solution may be just at the edge of the color transition pH for bromphenol blue. dilution with the sample, in buffer, should push the pH back up (a little) and the color should return to blue. if not then you can add a little higher pH buffer to the sample until it turns blue
2- some antibodies give less background with either milk or BSA. so we use one of the blocking agants that gives low background for a particular antibody. usually its milk but sometimes BSA as well.
TEMED appears yellow due to oxidation.