Qualitative Analysis of Product

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 • 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 causesseparation of molecules + ++= +

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

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

17. Uses for PAGE • Separate from other proteins • 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 molecularweight 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 Constituents1 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.