Posted 01 July 2004 - 09:18 PM
Posted 03 July 2004 - 04:14 PM
I would firstly establish another line of evidence for complex formation using gel filtration where an additional peak should be visible at 187kDa after the phosphorylation event- which can be demonstrated with either antibody.
This complex of the two proteins P1-P2 can be physically separated on a 15% SDS-PAGE and you can actually see the doublet if certain run conditions are met. Here is how.
Cast a 15% denaturing gel with just the separating gel-no stacking.
This gel must have a run length of at least 10 cm. Mini gels are out.
Use prestained markers
Run the electrophoresis until the 100kDa size marker is in the middle of the gel.
Transfer and blot with- 1. P1+ P2 antibody combo; 2. P1 Ab alone; 3. P2 Ab alone 4. Ab to phosphoamino acid (Sigma) and 5. Ab to P1 + Ab to phosphoaminoacid combo.
Posted 19 July 2004 - 02:27 AM