1. SDS PAGE Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis
Powerpoint from
modified 12/2007 CLK

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

3. 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?

4. Uses of SDS-PAGE Determine protein size Identify protein
Determine sample purity
Identify existence of disulfide bonds
Quantify amounts of protein
The fun video:

5. 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.
modified 12/2007 CLK

6. 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!

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

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

9. 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.

11. 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

12. 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.

13. 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

14. 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

15. 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

17. SDS page and sulfur bridges