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

2. 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)

5. 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

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

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

8. 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

10. 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.

12. 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

14. 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

15. 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.

16. 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

17. 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