Page electrophoresis 08_aug12

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Published on March 23, 2014

Author: prabhakarareddyav

Source: slideshare.net

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 • 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

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 +

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 =

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

Horizontal Gels • Gel Box set up frequently used in DNA analysis

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

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

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

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

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

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

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

Molecular Weight vs Distance

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

SDS Polyacrylamide Electrophoresis

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

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

% 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

Vertical Polyacrylamide Gel Electrophoresis

Equipment for Electrophoresis

Gel Electrophoresis Equipment Mini-PROTEAN Tetra Cell

Closed Mini Gel holder

Open Gel Holder: Allows New Gel to be Inserted

Gel Holders Placed in Mini-Protean Tetra Cell

Procedure in Short LoadGe Place Buffer Equip

Electrophoresis of Samples Setting Up and Running Mini-PROTEAN TGX Precast Gels – Youhttp://www.youtube.co 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

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