Protein Electrophoresis & Western Blotting Biotechniques (BIOL 410) Protein Electrophoresis & Western Blotting

Polyacrylamide Gel Different than agrose Proteins do not have a constant net change Charges vary across the molecule, and with shape, affecting Electrophoretic mobility. Sodium Sodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS PAGE Polyacrylamide gel is hydrophilic medium Like agarose pore size can be regulated by concentration Preparation methods can also vary pore size, so we will be using pre-cast gels Polyacrylamide gels are usually 250um thick By modifying pH, and using salt buffers, we can get proteins to a equal net change (Isoelectric point) Then making the proteins slightly negative gives them a equal charge

SDS PAGE The end result is the protein subunits being separated by size Kilo-Daltons (KD; mass)

Protein Gel Electrophoresis Like DNA Protein can be separate using a Gel solid phase Proteins will separate based on size

Once separated Once proteins have been sorted by size using electrophoresis, We can transfer to a more stable medium Stain for observation Transfer process is called western blotting. With anti-body staining; immunoblotting

Western Blot Uses an electrical current to transfer sorted proteins to Nitrocellulose paper More stable than a thin Polyacrylamide gel Easier to stain and record data Longer lived, can last months-years if stored properly.

Western Blotting Process Electrical current applies to get proteins to migrate laterally onto the nito cellulose paper Blotting apparatus Nitrocellulose is protein sensitive, mishandling could cause false results. Do not touch with bare hands Do not place directly on counter

Western Blotting History Developed in 1970’s George Stark (no relation to Tony Stark) Found nitrocellulose to bind electronegative molecules DNA & RNA Modified proteins with negative charge also bound

Western Blotting History A similar technique was being developed by Harry Towbin and Julian Gordon in Switzerland Both groups decided to collaborate, instead of compete Name is a play on the technique developed by Edward Southern in the 1960’s to transfer DNA to nitrocellulose paper The first application of immunoblotting.

Today’s Lab Prepare Protein samples from several closely related species Separate and denature myosin protein subunits using vertical gel electrophoresis Poly-Acridimide Gel While gel electrophresis is running examine DNA gels from thursday Set up Western Blott

Next Lab Remove nitrocellulose from blocking solution and stain for proteins Antibody staining (Immunoblotting) Observation and measurement of protein samples Measurement of proteins using Standard Curve Phylogenetic analysis