BIOL308 Lecture Notes - Lecture 16: Primary And Secondary Antibodies
Document Summary
Sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page and western blotting. Sds; negatively charged detergent; binds to hydrophobic protein regions - helps protein unfolding. Proteins bind lots of sds in constant ratio to their mass - sds gives them a negative charge proportional to their mass (equal charge density per unit length) - protein intrinsic charge is masked. Electrophoresis under denaturing conditions; proteins migrate towards the positive electrode when voltage is applied to a gel. Separate on the basis of molecular weight rather than intrinsic charge. Proteins detected by different stains: gel stained with coomassie brilliant blue, silver, etc. First step is sds page - proteins are denatured by heat and detergent (sds) and electrophoresed, separating on the basis of size. The proteins are transferred to a membrane - western blotting. The membrane is incubated with an antibody specific for one of the proteins.