Qualitative Analysis of Product

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Qualitative Analysis of Product Polyacrylamide Gel Electrophoresis

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Qualitative Analysis of Product. Polyacrylamide Gel Electrophoresis. Analysis of Product. Purity of product Different methods have different levels of detection Electrophoresis: Agarose and PAGE Demonstrates: Molecular Weight, Quantity, Purity, and identity. Electrophoresis. - PowerPoint PPT Presentation

Transcript of Qualitative Analysis of Product

Page 1: Qualitative Analysis of Product

Qualitative Analysis of Product

Polyacrylamide Gel Electrophoresis

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Analysis of Product

• Purity of product • Different methods have different levels of

detection • Electrophoresis: Agarose and PAGE• Demonstrates: Molecular Weight,

Quantity, Purity, and identity

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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

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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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V = v

Net charge on molecules determines its attraction to + or - electrode

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A voltage difference between either side of gel causes

separation of molecules

+

+

++=

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P

Polyacrylamide Gel Creates tunnels in gel for molecules to move through

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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

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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

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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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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

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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

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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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Horizontal Gels

• Gel Box set up frequently used in DNA analysis

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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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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

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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

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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

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SDS Polyacrylamide Electrophoresis

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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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Procedure in Short

LoadGe Place Buffer

Equip

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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.

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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.