1. Qualitative Analysis of Product
Polyacrylamide Gel Electrophoresis

2. Analysis of Product Purity of product
Different methods have different levels of detection
Electrophoresis: Agarose and PAGE
Demonstrates: Molecular Weight, Quantity, Purity, and identity

3. Electrophoresis Horizontal Agarose Gels Vertical Polyacrylamide Gels
Mainly used for DNA analysis
High sensitivity with ethidium bromide
Vertical Polyacrylamide Gels
Used for Protein analysis
Sensitivity with Coomasie Brilliant blue 50 ng
IEF electrophoresis
Western Blot technique

4. Electrophoresis and Movement of Molecules
Molecules can have distinct charges
Positive or Negative
Net charge will cause different movement through gel
Molecules can have different shapes
Linear
globular
Alpha helix +

5. Net charge on molecules determines its attraction to + or - electrode
V = v

6. A voltage difference between either side of gel causes separation of molecules+
++= +

7. Polyacrylamide Gel Creates tunnels in gel for molecules to move through

8. Principles of Electrophoresis
- Ohm’s Law : voltage is proportional to the current flow and inversely proportional to the resistance of the current flow
Voltage = current x resistance
Using direct current from power supply an electric potential is applied across the gel
This force results in charge movement through a gel matrix to its opposite charge

9. What is electrophoresis?
Forced migration of charged particles in an electric field Fel = Eq
q = charge, E= electric field
Molecules accelerate rapidly and are slowed by frictional forces
Electrophoretic mobility is determined as:
v = Eq / f f = friction coefficient
Mobility is intrinsic to the macromolecule and depends on frictional properties, charge

10. Macromolecular charge
Macromolecules have a variable net charge that depends on pH
pH at which net charge is zero = pI
Electrical shielding of charge occurs when counterions are solvated
V = V=

11. Protein in a salt solution
About Charge
Unlike isolated ions, such as Na + and Cl-, macromolecules have a variable net charge
Charge depends on pH
Counter ions provide electrical shielding
These effects can alter movement of macromolecules

12. PAGE
Native : Protein is prepared with little disturbance to its native form
Proteins can aggregate
Movement of samples through the gel can be inconsistent
SDS : Sodium Dodecyl Sulfate Is a detergent
Protein coated with a negative charge in proportion to its molecular weight
Denatures and unfolds protein
Added reducing agents (DTT) break disulfide bonds and tertiary structure

14. Agarose gels Usually used in DNA analysis
Made up of linear polysaccharide mol wt of 12,000
Basic repeating unit is agarobiose
Gels are prepared at 1% to 3% providing tunnels for molecules to move through
DNA can be much larger then most proteins

15. Horizontal Gels
Gel Box set up frequently used in DNA analysis

16. Agarose Gel with DNA Bands
markers
DNA is negatively charged
Smaller sized DNA moves faster than Larger DNA
Markers are used to determine relative sizes of DNA pieces

17. Uses for PAGE Separate from other proteins Determines
Proteins separated by size
Isoelectric point
Determines
Molecular size of protein
Quantifies the amount present
Displays Impurities
Used in western blot assays

18. Determine Molecular Weight
1. Run standard molecular weight markers
on gel
2. Run unknown protein on the same gel
3. Create a graph of the mol wt versus
distance molecule has moved
4. Using the distance the unknown has moved determine the molecular weight from graph

19. Molecular Weight Markers
Migration of molecular weight of standards are compared to unknown samplewt std vs unknown

20. Molecular Weight vs Distance

21. Western Blot Analysis

22. SDS Effect on Protein Movement
Sodium Dodecyl Sulfate denatures protein and covers it with negative charges : moves to + end
Vertical gels are designed so the top of the gel box is attached to the negative power outlet
The bottom of the gel box is attached to the positive power outlet
Movement through the PAGE gel is proportional to mass

23. SDS Polyacrylamide Electrophoresis

24. Movement of Proteins on an SDS Gel
Protein Migration -
Stacking of proteins at top of gel at start
Highest Molecular Wt. protein
Distribution of proteins in a charged field +
Low weight molecular dye

25. % Polyacrylamide in Gel
Gels can be made at different concentrations of polyacrylamide
Example: gels made at 3%,6%,9% and 12% will produce different openings through which the molecule will migrate
The larger the opening allows large molecules to move through the gel

26. Vertical Polyacrylamide Gel Electrophoresis

27. Equipment for Electrophoresis

28. Procedure in Short
LoadGe Place Buffer
Equip

29. Electroporhesis of Samples
1 part Protein Sample: 1 part Laemmli Buffer are boiled in Eppendorf tube
Set up SDS-PAGE electrophoresis (or gel) box by SOP
Place 25ul of boiled sample: loading buffer into gel wells
Run at 75 mamp for 1-2 hours
Remove, stain with Coomassie blue and destain with DI water.

30. Laemmli Buffer Constituents 1 part Protein Sample: 1 part Laemmli Buffer
BME (beta-mercaptoethanol) and/or DTT (dithiothreitol) are reducing agents that break disulfide bonds causing proteins to go from tertiary to secondary structure.
SDS (sodium dodecyl sulfate) is a detergent (soap) that breaks delicate hydrogen bonds in the protein causing proteins to go from secondary to primary structure and puts negative charges all over the protein surface.
Proteins are pulled downwards through the gel to the anode or positive pole proportional to their mass or MW.
Broomophenol blue is an indicator dye that moves ahead (or in front) of most of the proteins in the samples.
Glycerol increases the density of the proteins in a sample so that the proteins will fall to the bottom of the well, minimizing their loss.