ELECTROPHORETIC METHODS

1. It is the process of moving charged biomolecules in solution by applying an electrical field across the mixture.

2. Biomolecules moved with a speed dependent on their charge, shape, and size and separation occures on the basis of molecular size.

Electrophoresis is used: for analysis and purification of very large molecules (proteins, nucleic acids)for analysis of simpler charged molecules (sugars, amino acids, peptides, nucleotides, and simpler ions).

Basic principles of electrophoresis

When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.

1. Factors influenced electrophoresis mobility:

2. net charge of the molecule

3. size and shape

4. concentration of the molecule in solution

Amino acids have characteristic titration curves

Fully protonated form at wery low pH

At the midpoint – pK1=2.34 there is equimolar concentration of proton donor and proton acceptor.

+

Dipolar ion

+At the midpoint – pK=9.60 there is equimolar concentration of proton donor and proton acceptor.

Proton donor

Proton acceptor

Proton donor

Proton acceptor

Izoelectric point

Adopted from: D.L. Nelson, M.M. Cox Lehninger Principle of Biochemistry

Electrophoresis is carried out by applying a thin layer

Aqueous protein solution is immobilized in a solid hydrophilic support.

Solid matrix with pores which are used:• paper• starch• cellulose acetate• polyacrylamide• agar/agarose

Molecules in the sample move through porous matrix at different velocity.

• Electrophoresis can be one dimensional (i.e. one plane of separation) or two dimensional.

• One dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two dimensional separation of proteins is used for finger printing , and when properly constructed can be extremely accurate in resolving all of the proteins present within a cell (greater than 1,500).

• Most common stabilizing media are polyacrylamide or agarose gels.

• Function of buffer1. carries the applied current

2. established the pH

3. determine the electric charge on the solute

• High ionic strength of buffer– produce sharper band– produce more heat

• Commonly used buffer• Barbital buffer & Tris-EDTA for protein

• Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8)

Buffers

Zone electrophoresis

• Much simple method• Much greater resolution• Require small sample• Acetate cellulose – support medium

Protein separation depends on :• Type and number of ionizable side chains of amino acids - R.• Size of net charge (positive or negative).

• Negatively charged proteins move towards the anode.• Positively charged proteins move towards the cathode.

Stripe of cellulose acetate

Electrophoresis

Major protein componentsseparate into discrete zones

Densitometer tracing density of zones is proportional to the amount of protein

Example of application of zone electrophoresis in clinical practice

Hypergamaglobulinemia

Hypogamaglobulinemia

Normal serum

• Gel is a colloid in a solid form (99% is water).

• Gel material acts as a "molecular sieve.

• During electrophoresis, macromolecules are forced to move through the pores when the electrical current is applied.

Gel electrophoresis

• Agarose and polyacrylamide gels are across-linked, spongelike structure

• It is important that the support media is electrically neutral. Presence of charge group may cause:-Migration retardation

-The flow of water toward one or the other electrode so called ‘Electroendosmosis (EEO)’, which decrease resolution of the separation

Support media

Agarose – highly purified polysaccharide derived from agar (extracted from seeweed), long sugar polymers held together by hydrogen and hydrophobic bonds.

Acrylamide (CH2=CH-CO-NH2)Polyacrylamide gel structure held together by covalent cross-links

• For the separation of (1) large protein or protein complex (2) polynucleotide 50-30,000 base-pairs

• The pore size is determined by adjusting the concentration of agarose in a gel (normally in the rank of 0.4-4%

Agarose gels

OH

O OH

CH2OHO

O OHO

O

O

CH2=CHCONH2 + CH2(NHCOHC=CH2)2Acrylamide N,N,N,N-methylenebisacrylamide

Free radical catalyst

-CH2-CH-CH2-CH-CH2-CH-

-CH2-CH-CH2-CH-CH2-CH-

CONHCH2NHCO

CONHCH2NHCO

CONH2

NH2CO

n

n

Polyacrylamide gels

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

• SDS (also called lauryl sulfate) - anionic detergent • Molecules in solution with SDS have a net negative charge within a wide pH

range. • A polypeptide chain binds amounts of SDS in proportion to its relative

molecular mass. • The negative charges on SDS destroy most of the complex structure of

proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field.

Diagrams of vertical slab gel assembly

Determination of molecular mass

Stain Detection limit

Ponceau S 1-2 g

Amido Black 1-2 g

Coomassie Blue 1.5 g

India Ink 100 ng

Silver stain 10 ng

Colloidal gold 3 ng

Commonly used protein stains

Staining with Coomasie blue

1 2 3

1

2

3

Assesment of individual lines

An ethidium-stained gel photographed under UV light

**Each band that you see is a collection of millions of DNA molecules, all of the same length!!

Western blott technique

• Western blot (also called immunoblot) is a technique to detect specifically one protein in a mixture of large number of proteins and to obtain information about the size and relative amounts of the protein present in different samples.

• In first proteins are separated using SDS-polyacrylamide gel electrophoresis.

• Then they are moved onto a nitrocellulose membrane. The proteins retain the same pattern of separation they had on the gel.

• An antibody is then added to the solution which is able to bind to its specific protein and forms an antibody-protein complex with the protein of interest. (In fact there is no room on the membrane for the antibody to attach other than on the binding sites of the specific target protein).

• Finally the nitrocellulose membrane is incubated with a secondary antibody, which is an antibody-enzyme conjugate that is directed against the primary antibody.

• The location of the antibody is revealed by incubating it with a substrate that the attached enzyme converts to a product that can be seen and followed and then photographed.

Isoelectric focusation

Proteins are separated in pH gradient.Protein migrate into the point where its net charge is zero – isoelectric pH.Protein is positively charged in solutions at pH values below its pI.Protein is negatively charged in solution at pH above its pI.

Two-dimensional gel electrophoresis(2-D electrophoresis )

In the first dimension, proteins are resolved in according to their isoelectric points (pIs) using immobilized pH gradient electrophoresis (IPGE), isoelectric focusing (IEF), or non-equilibrium pH gradient electrophoresis. In the second dimension, proteins are separated according to their approximate molecular weight using sodium dodecyl sulfate poly-acrylamide-electrophoresis (SDS-PAGE).

Electrophoreogram of the mixture of proteins

Protein „maps“ are compare with control pattern of normal healthy person and abnormalities are analysed

Capillary electrophoresis

Capillaries are typically of 50 µm inner diameter and 0.5 to 1 m in

length. Due to electroosmotic flow, all sample components migrate towards the negative electrode. The capillary can also be filled with a gel, which eliminates the electroosmotic flow. Separation is accomplished as in conventional gel electrophoresis but the capillary allows higher resolution, greater sensitivity, and on-line detection.

Electroosmotic flow

The surface of the silicate glass capillary contains negatively-charged functional groups that attract positively-charged counterions. The positively-charged ions migrate towards the negative electrode and carry solvent molecules in the same direction. This overall solvent movement is called electroosmotic flow. During a separation, uncharged molecules move at the same velocity as the electroosmotic flow (with very little separation). Positively-charged ions move faster and negatively-charged ions move slower.