1. Quality Control of Product
Polyacrylamide Gel Electrophoresis

2. Analysis of Product Quality Control involves the entire process of
obtaining a product that meets defined
specifications expressing both its purity and
potency
Testing methods include cell biology, virology,
chemistry, analytical chemistry, molecular
biology & the potency of the product
Different methods have different levels of detection ie, values can go from grams to nanograms

3. 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. 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=

6. Electrophoresis Horizontal Agarose Gels
Agarose forms a gel or molecular sieve that supports the movement of small materials in solution used for DNA
Vertical Polyacrylamide Gels
Made of Polyacrylamide
Used for Protein molecular size, shape, charge
IEF electrophoresis
Western Blot technique

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

8. 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

9. 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

10. PAGE
Native : Protein is prepared with little disturbance to the cellular material
Proteins are associated
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
Reducing agents (DTT)break amino acid cross-links

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

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

14. 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

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

16. Molecular Weight vs Distance

17. Western Blot Analysis Identifies protein through antibody interaction
Run proteins on denatured gel (SDS-PAGE)
Transfer (blot) proteins onto membrane
Probe the membrane with primary antibody
Add secondary antibody (this antibody is linked to an enzyme)
Substrate is added and color appears

18. SDS Polyacrylamide Electrophoresis

19. 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 not to charge

20. 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

21. % 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

22. Vertical Polyacrylamide Gel Electrophoresis

23. Equipment for Electrophoresis

25. Gel Electrophoresis Equipment Mini-PROTEAN Tetra Cell

26. Closed Mini Gel holder

27. Open Gel Holder: Allows New Gel to be Inserted

28. Gel Holders Placed in Mini-Protean Tetra Cell

29. Procedure in Short
LoadGe Place Buffer
Equip

30. Electrophoresis of Samples
Setting Up and Running Mini-PROTEAN TGX Precast Gels – Youhttp:// m/watch?v=XnEdmk1SqvgT ube
Samples: boiled 3’ with loading dye (2x Laemmli buffer + running dye) Mini-PROTEAN tetra cell: Set up according to SOP given in workbook Power settings: 75 volts for 45 – 60 minutes Running dye should not run off the bottom of gel