MB 7112: Analysis of protein Purity by Electrophoresis

Electrophoresis is the migration of charged molecules in solution in response to an electric field. The rate of migration depends on: the strength of the field (Voltage); the net charge, size and shape of the molecules The ionic strength, viscosity and temperature of the medium in which the molecules are moving.

Electrophoresis is a technique used to separate molecules based on their size and charge, according to the following equation

where v = the rate (velocity) of migration, E = strength of the electrical field, z = charge on the molecule and = frictional force on the molecule. =6r where is the viscosity of the medium and r is the stokes radius of the molecule.

Electrophoresis
As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used to Study the properties of a single charged species, and as a separation technique.

Electrophoresis
The sample is run in a support matrix e.g. paper, cellulose acetate, starch gel, agarose gel polyacrylamide gel. At the end of the run, the matrix can be stained and used for scanning, autoradiography or storage.

Agarose and polyacrylamide also separate molecules by size Dilute agarose gels are generally more rigid and easy to handle at low concentration, used to separate larger macromolecules Nucleic acids, Large proteins and protein complexes. A polyacrylamide gel contains long linear polymers of acrylamide cross-linked to each other by bis-acrylamide. It is easier to handle at higher concs, used to separate most proteins and small oligonucleotides

Proteins vs Nucleic Acids Proteins are amphoteric, their net charge determined by the pH of the medium. The net charge carried by a protein is independent of its size, the charge carried per unit mass differs from protein to protein. At a given pH under non-denaturing conditions, the electrophoretic separation of proteins is determined by size and charge of the molecules.

Proteins vs Nucleic Acids Nucleic acids however, Remain negative at any pH used for electrophoresis and Carry a fixed negative charge per unit length of molecule, provided by the PO4 group of each nucleotide of the nucleic acid. Electrophoretic separation of nucleic acids is strictly according to size.

Polyacrylamide gels
Started as disc elerctrophphoresis Then thin slab electrophoresis

Polyacrylamide gels
Started as disc elerctrophphoresis Then thin slab electrophoresis

Advantages Less heat generated during electrophoresis Easy diffusion of dyes during staining/destaining

Simple/native PAGE
Done at pH where protein remains stable in native form Mobility dependent on both charge and size

pH range 8-9 so that its ve

What about basic proteins? use buffer of lower pH Cathode at bottom of the gel

Simple/native PAGE
Acrylamide and Methylene Bisacrylamide (polylinker)

1 part crosslinker : 20-50 parts monomer

Total % of acrylamide determines pore size 3-4% for 100kDa proteins 20% for 10kDa proteins Initiate polymerisation with 1.5-2mM APS and TEMED Polymerisation within 30min at room temp

SDS-PAGE: Separation of Proteins under Denaturing conditions SDS an anionic detergent Denatures proteins by specific binding in a mass ratio of 1.4:1. SDS confers a negative charge to the polypeptide in proportion to its length It is usually necessary to reduce disulphide bridges in the proteins 2- mercaptoethanol or dithiothreitol. In denaturing SDS-PAGE separations therefore, migration is determined by molecular weight.

Denaturation ensures that the proteins no longer have any secondary, tertiary or quaternary structure

SDS-PAGE separates proteins based on their primary structure or size but not the amino acid sequence!

For most applications, the ratio of crosslinker (bis-acrylamide) to monomer (acrylamide) is kept at 37.5 to 1. The gel pore size is then dependent on the total acrylamide concentration.
% Acrylamide
7.5 10 12 15

Size Range of Molecules

45,000 - 400,000 22,000 - 300,000 13,000 - 200,000 2,500 - 100,000

SDS-PAGE Standards Phosphorylase B Serum albumin Ovalbumin Carbonic Anhydrase Trypsin Inhibitor 97,400 66,200 45,000 29,000 21,500

MW

Lysozyme

14,400

Continuous and Discontinuous Buffer Systems

A continuous system has

only a single separating gel and uses the same buffer in the tanks and the gel. In a discontinuous system, a non-restrictive large pore gel, (the stacking gel) is layered on top of a separating (resolving) gel. Each gel is made with a different buffer, the tank buffers are different from the gel buffers. The resolution obtained in a discontinuous system is much better

Determination of Molecular Weight

SDS-PAGE of proteins of interest alongside those of known molecular weight A linear relationship between the log of the molecular weight of an SDS-denatured polypeptide and its Rf.
The Rf is the ratio of the distance migrated by the molecule to that migrated by a marker dye-front.

To determine the relative molecular weight by electrophoresis (Mr),

Plot a standard curve of distance migrated vs. log10MW for known samples, Read off the logMr of your sample after measuring distance migrated on the same gel.

Mass spectrophotometry is the other method (look it up!!)

MB 701: Electrophoresis

Urea Gels
Suitable for proteins that are insoluble at the low ionic strength characteristic of electrophoretic conditions

6-8M urea is used to denature the protein

Mecarptoethanol used to disrupt disulphide bonds Proteins separated according to charge and subunit size

Gradient gels
Used to separate native proteins Separation by size only

Acrylamide concetration varies 3% at top to 30% at bottom

Electrophoresis is done at high pH Protein moves up to the thickness that prevents further movement

Useful for determination of molecular weight

Isoelectric focusing
Gels run in a pH gradient Protein moves until it reaches its pI 5-6% acrylamide or 2% agarose used The gel is used as a stabilizing medium rather than a filter Pre-cast gels more commonly used

Two Dimensional (2-D) electrophoresis

Two dimensions analysis included in the same

Isoelectric focusing (pH gradient)

Sub-unit size (SDS-PAGE) Yields up to 1000 individual protein spots Spot of interest can be cut out and identified Used for crude cell extracts

Applicable for studying expression

A 2-D gel yields spots that can be excised for sequencing