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Quality Control of Product Polyacrylamide Gel Electrophoresis

Quality Control of Product

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Quality Control of Product. Polyacrylamide Gel Electrophoresis. Analysis of Product. Quality Control involves the entire process of obtaining a product that meets defined specifications expressing both its purity and potency - PowerPoint PPT Presentation

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Page 1: Quality  Control of  Product

Quality Control of Product

Polyacrylamide Gel Electrophoresis

Page 2: Quality  Control of  Product

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

Page 3: Quality  Control of  Product

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

+

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Page 5: Quality  Control of  Product

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 =

Page 6: Quality  Control of  Product

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

Page 7: Quality  Control of  Product

Horizontal Gels

• Gel Box set up frequently used in DNA analysis

Page 8: Quality  Control of  Product

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

Page 9: Quality  Control of  Product

Agarose Gel with DNA Bands

• DNA is negatively charged

• Smaller sized DNA moves faster than Larger DNA

• Markers are used to determine relative sizes of DNA pieces

markers

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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

Page 11: Quality  Control of  Product

P

Polyacrylamide Gel Creates tunnels in gel for molecules to move through

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Uses for PAGE

• Separates proteins from each other– 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

Page 14: Quality  Control of  Product

Determine Molecular Weight

1. Run standard molecular weight markers on gel2. Run unknown protein on the same gel3. Create a graph of the mol wt versus distance molecule has moved4. Using the distance the unknown has moved

determine the molecular weight from graph

Page 15: Quality  Control of  Product

Molecular Weight Markers

Migration of molecular weight of standards are compared to unknown samplewt std vs unknown

Page 16: Quality  Control of  Product

Molecular Weight vs Distance

Page 17: Quality  Control of  Product

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

Page 18: Quality  Control of  Product

SDS Polyacrylamide Electrophoresis

Page 19: Quality  Control of  Product

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

Page 20: Quality  Control of  Product

Movement of Proteins on an SDS Gel

Stacking of proteins at top of gel at start

Low weight molecular dye

-

+

Distribution of proteins in a charged field

Protein Migration

Highest Molecular Wt. protein

Page 21: Quality  Control of  Product

% 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

Page 22: Quality  Control of  Product

Vertical Polyacrylamide Gel Electrophoresis

Page 23: Quality  Control of  Product

Equipment for Electrophoresis

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Gel Electrophoresis EquipmentMini-PROTEAN Tetra Cell

Page 26: Quality  Control of  Product

Closed Mini Gel holder

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Open Gel Holder:Allows New Gel to be Inserted

Page 28: Quality  Control of  Product

Gel HoldersPlaced in Mini-Protean Tetra Cell

Page 29: Quality  Control of  Product

Procedure in Short

LoadGe Place Buffer

Equip

Page 30: Quality  Control of  Product

Electrophoresis of Samples

Setting Up and Running Mini-PROTEAN TGX Precast Gels –

Youhttp://www.youtube.com/watch?v=XnEdmk1SqvgTube

Samples: boiled 3’ with loading dye (2x Laemmli buffer + running dye) Mini-PROTEAN tetra cell: Set up according to SOP given in workbookPower settings: 75 volts for 45 – 60 minutesRunning dye should notrun off the bottom of gel