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

• 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

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

+

V = v

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

A voltage difference between either side of gel causes

separation of molecules

+

+

++=

P

Polyacrylamide Gel Creates tunnels in gel for molecules to move through

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

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

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 =

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

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

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

Horizontal Gels

• Gel Box set up frequently used in DNA analysis

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

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

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

Molecular Weight Markers

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

Molecular Weight vs Distance

Western Blot Analysis

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

SDS Polyacrylamide Electrophoresis

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

Procedure in Short

LoadGe Place Buffer

Equip

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.

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.