Chromatofocusing is a protein separation technique that uses ion exchange resins and buffers with changing pH to separate proteins based on their isoelectric point. As the buffer pH passes through a protein's pI, the protein loses its charge and elutes from the resin. Chromatofocusing provides high resolution separation of proteins that have similar pI values. However, some proteins may aggregate at high concentrations and clog the resin. Isoelectric focusing uses immobilized pH gradients in gels to separate proteins based on their pI through electrophoresis. Two-dimensional electrophoresis separates proteins first by pI using isoelectric focusing, then by molecular weight using SDS-PAGE to provide high resolution separation and identification of

1. Analytical and Biochemical
Techniques

2. Chromatofocusing
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).

3. 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.

13. 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

14. 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

15.  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.

17. • 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 Gawish
17

18. PREPARATION OF IEF GEL
Carrier ampholytes (suitable pH) and riboflavin
mixed with acrylamide solution
Mixture is poured over a glass plate which
contain spacer
Second glass plate is placed on first
Gel is polymerised

19. This takes 2-3 hr
After the gel has set glass plates are prised apart
Electrode wicks are laid along the long length of
each side of the gel
Potential difference is applied
Ampholytes form a pH gradient between anode
and cathode

20. The power is then turned off
Samples applied by laying on gel filter paper soaked in
the sample
Voltage is again applied for 30 min
Proteins having positive charge will migrates towards the cathode.
negatively charged protein will migrates towards anode
Become stationary when they reaches isoelectric point

22. The gel is washed with trichloroacetic acid
This precipitaes the proteins and allows smaller
ampholytes to be washed out
Gel is stained with Coomasie Brilliant Blue
Destained

23. A TYPICAL ISOELECTRIC FOCUSING GEL

24. Two Dimensional Electrophoresis
(2-DE)

25. Three properties of proteins
 Size: molecular weight (utilized in 2-DE)
 Charge: pI (utilized in 2-DE)
 Hydrophobicity

26. What is 2-DE?
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
Digest to peptide fragment MS analysis

27. Two dimensional electrophresis, 2-DE
 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.

28. Evolution of 2-DE methodology
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.
In the past

29. Evolution of 2-DE methodology
Problems with traditional 1st dimension IEF
 Works well for native protein, not good for denaturing
proteins, because:
OPERATOR DEPENDENT
 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.

30. Evolution of 2-DE methodology
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)

31. Run 2-DE, a quick overview

33. Run 2-DE step by step

34. Run 2-DE step by step

35. Total E. coli Proteins - 2-Dimensional Gel

38. 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.