Exploring Proteins with SDS-PAGE and Gel Electrophoresis Techniques

1. Chemistry 4010 Lab • It’s all about PROTEINS…

2. SDS-PAGE • Understanding SDS and PAGE • SDS- Sodium Dodecyl Sulfate • a detergent soap that dissolves hydrophobic molecules • also has a negative charge • by incubating the cell with SDS, the membranes are dissolved and proteins are solubilized • protein now possesses negative charges • PAGE- Polyacrylamide Gel Electrophoresis

4. SDS-PAGE • Understanding SDS and PAGE • SDS- Sodium Dodecyl Sulfate • PAGE- Polyacrylamide Gel Electrophoresis • polymer of acrylamide monomers • with a mixture of other buffers and solutions this polymer can be changed into a gel matrix • electricity is used to pull the protein through the gel • the negatively charged protein moves to the positive poles

5. Side view

6. Top View

7. How does it all work • add the proteins to the gel matrix • turn on the current • negatively charged proteins will move through the gel • smaller sized proteins will move at a faster rate due to their ability to maneuver through the gel

8. Proteins Moving through the Gel

9. What will the gel look like • smaller proteins will move through the gel faster while larger proteins move at a slower pace • Visualize the bands by staining the gel with Coomassie Blue

10. SDS- PAGE Setup

11. SDS-PAGE vs. Gel Electrophoresis • SDS-PAGE is a great tool for analyzing proteins, whereas Gel Electrophoresis is used to study DNA.

12. Gel Electrophoresis • Makes use of an agarose gel • Agarose is a linear polysaccharide (average molecular mass about 12,000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose. Agarose is usually used at concentrations between 1% and 3%.

13. Gel Electrophoresis • DNA is cut by restriction enzymes to yield many different sized pieces of DNA • Like SDS-PAGE, an electric current is applied and the negatively charged DNA moves through the gel • Smaller pieces move faster

14. Gel Electrophoresis Setup

15. THEORYSDS-PAGE & Gel Electrophoresis • These techniques make use of the fact that molecules are being separated based on size and charge, based on the following equations • v = the rate (velocity) of migration • E = the strength of the electrical field • z = the charge on the molecule • f = the frictional force on the molecule • Frictional force can then be defined as • η is the viscosity of the medium • r is the stokes radius of the molecule

16. Gel Filtration or GPC • Gel filtration chromatography is a separation based on size • stationary phase consists of porous beads with a well-defined range of pore sizes • mobile phase consists of the solvent

17. GPC • Proteins that are too large to fit inside any of the pores-EXCLUDED • access only to the mobile phase between the beads and elute first. • Proteins of intermediate size –PARTIALLY INCLUDED • fit inside some but not all of the pores in the beads. These proteins will then elute between the large ("excluded") and small ("totally included") proteins • Proteins that are small enough can fit inside all the pores in the beads -INCLUDED • access to the mobile phase inside the beads as well as the mobile phase between beads and elute last

18. Theory of GPC • These separations can be described by this equation • Vr = the retention volume of the protein • Vo = the volume of mobile phase between the beads of the stationary phase inside the column (sometimes called the void volume) • Vi = the volume of mobile phase inside the porous beads (also called the included volume) • K = the partition coefficient (the extent to which the protein can penetrate the pores in the stationary phase, with values ranging between 0 and 1).

19. GPC Setup

20. Conclusion • All of these techniques serve to enhance our ability to better elucidate the size, charge and polarity of polymers in science • Understanding the parallel nature of these experiments will allow us to bridge the gap between natural and synthetic polymers