Page electrophoresis 08_aug12
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Transcript of Page electrophoresis 08_aug12
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Quality Control of Product
Polyacrylamide Gel Electrophoresis
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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
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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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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 =
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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
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Horizontal Gels
• Gel Box set up frequently used in DNA analysis
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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
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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
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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
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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
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Molecular Weight Markers
Migration of molecular weight of standards are compared to unknown samplewt std vs unknown
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Molecular Weight vs Distance
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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
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SDS Polyacrylamide Electrophoresis
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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
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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
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% 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
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Vertical Polyacrylamide Gel Electrophoresis
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Equipment for Electrophoresis
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Gel Electrophoresis EquipmentMini-PROTEAN Tetra Cell
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Closed Mini Gel holder
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Open Gel Holder:Allows New Gel to be Inserted
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Gel HoldersPlaced in Mini-Protean Tetra Cell
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Procedure in Short
LoadGe Place Buffer
Equip
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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