Analytical and Biochemical Techniques

Chromatofocusing is a protein-separation technique that allows

resolution of single and other ampholytes from a complex mixture

according to differences in their isoelectric point. Chromatofocusing

utilizes ion exchange resins and is typically performed on fast protein

liquid chromatography (FPLC) or similar equipment capable of

producing continuous buffer gradients though this is not a requirement.

In contrast to typical ion exchange chromatography, where bound

molecules are eluted from the resin by increasing the ionic strength of

the buffer environment, chromatofocusing elutes bound species by

altering the pH of the buffer. This changes the net surface charge of

bound molecules, altering their affinity for the resin. As the changing

pH of the buffer system traverses the pI of a given molecule, that

molecule will elute from the resin as it will no longer possess a net

surface charge (a requisite for molecular binding to ion exchange

resins).

Chromatofocusing

Chromatofocusing is a powerful purification technique with

respect to proteins as it can resolve very similar species only

differing by 0.02 pH units that may not separate well, or at all,

using traditional ion exchange strategies.

A major drawback to this technique is that some proteins will

aggregate when present at relatively high concentrations and

carry no net surface charge. This can cause blockage of the

resin, which is highly problematic when using sealed columns

of ion exchange resin on FPLC equipment, resulting in pressure

build up and possible equipment failure.

Apparent aggregation issues can sometimes be overcome by

limiting the sample concentration and use of buffer additives

that prevent aggregate formation.

ISOELECTRIC FOCUSING

Electrophoretic method that separates

proteins according to the iso-electric points

Is ideal for seperation of amphoteric

substances

Seperation is achieved by applying a potential

difference across a gel that contain a pH

gradient

Isoelectric focusing requires solid support

such as agarose gel and polyacrylamide gel

Isoelectric focusing gels contain synthetic

buffers called ampholytes that smooth the pH

gradients.

Ampholytes are complex mixtures of

synthetic polyamino-polycarboxylic acids

Commercially available ampholytes are-

BIO-LYTE

PHARMALYTE

It gives good separation with a high resolution compared to any other method

Resolution depends on

I. The pH gradient,

II. The thickness of the gel

III. Time of electrophoresis,

IV.The applied voltage,

V. Diffusion of the protein into the gel.

• At pH = pI, a protein will have no net charge stop moving– At any other pH in the gradient, the protein has either a positive

charge (pH<pI) or negative charge (pH>pI)

• Runs requires higher voltages and longer periods of time, but gives resolution up ±0.001 pH

Dr Gihan Gawish17

PREPARATION OF IEF GEL

Gel is polymerised

Second glass plate is placed on first

Mixture is poured over a glass plate which contain spacer

Carrier ampholytes (suitable pH) and riboflavin mixed with acrylamide solution

Ampholytes form a pH gradient between anode and cathode

Potential difference is applied

Electrode wicks are laid along the long length of each side of the gel

After the gel has set glass plates are prised apart

This takes 2-3 hr

Become stationary when they reaches isoelectric point

Proteins having positive charge will migrates towards the cathode. negatively charged protein will migrates towards anode

Voltage is again applied for 30 min

Samples applied by laying on gel filter paper soaked in the sample

The power is then turned off

Destained

Gel is stained with Coomasie Brilliant Blue

This precipitaes the proteins and allows smaller ampholytes to be washed out

The gel is washed with trichloroacetic acid

A TYPICAL ISOELECTRIC FOCUSING GEL

Two Dimensional Electrophoresis (2-DE)

Three properties of proteins

Size: molecular weight (utilized in 2-DE)

Charge: pI (utilized in 2-DE)

Hydrophobicity

What is 2-DE?

Digest to peptide fragment MS analysis

1. First dimension:denaturing isoelectric focusing separation according to the pI

2. Second dimension:SDS electrophoresis (SDS-PAGE)Separation according to the MW

Interested spot

Only “Proteomics” is the large-scale screening of the proteins of a cell, organism or biological fluid, a process which requires stringently controlled steps of sample preparation, 2-D electrophoresis, image detection and analysis, spot identification, and database searches.

The core technology of proteomics is 2-DE

At present, there is no other technique that is capable of simultaneously resolving thousands of proteins in one separation procedure.

Two dimensional electrophresis, 2-DE

Traditional IEF procedure:

IEF in run in thin polyacrylamide gel rods in glass or plastic tubes.

Gel rods containing: 1. urea, 2. detergent, 3. reductant, and 4. carrier ampholytes (form pH gradient).

Problem: 1. tedious. 2. not reproducible.

Evolution of 2-DE methodology

In the past

Problems with traditional 1st dimension IEF

Works well for native protein, not good for denaturing proteins, because:

Takes longer time to run.

Techniques are cumbersome. (the soft, thin, long gel rods needs excellent experiment technique)

Batch to batch variation of carrier ampholytes.

Patterns are not reproducible enough.

Lost of most basic proteins and some acidic protein.

Evolution of 2-DE methodology

OPERATOR DEPENDENT

Resolution for IEF: Immobilized pH gradients.

Developed by Bjellqvist (1982, Biochem. Biophys Methods, vol 6, p317)

PH gradient are prepared by co-polymerizing acrylamide monomers with acrylamide derivatives containing carboxylic and tertiary amino groups.

The pH gradient is fixed, not affected by sample composition.

Reproducible data are presented.

Modified by Angelika Gorg by using thin film to support the thin polyacrylamide IEF gel, named Strips. (1988, Electrophoresis, vol 9, p 531)

Evolution of 2-DE methodology

Run 2-DE, a quick overview

Run 2-DE step by step

Run 2-DE step by step

Total E. coli Proteins - 2-Dimensional Gel

2-DE gel images of serum glycoprotein samples from the healthy and LC patients.

(A) Normal sample, (B) LC sample. The identified protein spots: (1) Anti TNFα antibody light chain (ATAL); (2) Chain L, structure of Fab D3h44 (D3h44); (3) Transthyretin (TTR); (4) AIM/CD69; (5) Alpha1-Antitrypsin (AAT); (6) Alpha2-HS-glycoprotein (AHSG) (7) Complement C3; (8) Zinc-alpha2-glycoprotein (ZAG); (9) Haptoglobin alpha2 chain (HpA2); (10) Ig heavy chain mu (BOT); (11) IGHM protein.