SDS PAGE Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis Powerpoint from www.colorado.edu/mcdb/MCDB3140/misc/Lab6.ppt modified 12/2007 CLK

What is SDS-PAGE? Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis A procedure to separate proteins and determine their Molecular Weights.

Steps in SDS-PAGE Extract Protein Solubilize and Denature Protein Separate Proteins on a gel Stain proteins (visualization) Analyze and interpret results What does it mean to denature a protein?

Uses of SDS-PAGE Determine protein size Identify protein Determine sample purity Identify existence of disulfide bonds Quantify amounts of protein The fun video: http://www.molecularstation.com/science-videos/video/33/demystifying-sds-page/

Electrophoretic Theory Charged molecules move in an electric field. Positive molecules move towards negative pole Negative molecules move towards positive pole. What would happen if you placed a protein in an electric field? Unpredictable – some are negatively, some positive, some neutral! Plus– They have widely different shapes. What do you know about protein? What happens to the uncharged proteins? SDS vs isoelectric focussing. www.colorado.edu/mcdb/MCDB3140/misc/Lab6.ppt modified 12/2007 CLK

What is so special about SDS? SDS is a negatively charged detergent. ‘Masks’ charge on protein so that all proteins act the same as regards charge. Break hydrogen bonds and unfold protein. Disrupts secondary and tertiary protein structures Prevents protein shape from influencing gel run. Prevents protein aggregation. Must have a major role since the technique is named after it!

ß-mercaptethanol disrupts disulfide bonds Notice that ß-mercaptethanol drives this reaction backwards

Protein gels are different Vertical Thinner Need SDS and β-mercaptethanol

Determine protein size Identify protein The smaller proteins move faster: proteins are unraveled by SDS so shapes are similar protein charges are swamped out by SDS charges But relationship is NOT linear.

                                             

Semi-log graphing Used when the relationship is exponential not linear. Note that the spaces between lines are not even! Each bold line represents a power of 10 101 100 102 103

Semi-log graphing Each bold line represents a power of 10 The faint lines between are multiples of that power of 10 You can choose which powers of 10 to use 103 102 101 100 The x axis is linear and all the spaces are the same.

Trend line is exponential, but on this type of graph it appears linear Well, not really. Your graph won’t be this pretty. We want to use the linear range. You will use Rf for your graph. Rf = sample distance traveled/ dye front distance traveled

Kaleidoscope Standard Protein Color MW (daltons) on Tris HCl gel Myosin Blue 204,649 B-galactosidase Magenta 127,511 Bovine serum albumin Green 85,130 Carbonic anhydrase Violet 37,830 Soybean trypsin inhibitor Orange 30,906 Lysozyme Red 27,230 Aprotinin 6,638

Western Blotting Combines resolution of SDS-PAGE with specificity of antibody detection. What you can find out: Presence of an antigen Amount of an antigen present Molecular Weight of antigen Allows for the identification of a single protein in a complex mixture

SDS page and sulfur bridges