Protocol:Isoelectric focussing electrophoresis

Isoelectric focusing is the electrophoretic method that separates proteins according to the iso-electric points. The net charge of a protein is the sum of all negative and positive charges of its amino-acid chains and their ionisable groups (amino and carboxyl termini). The isoelectric point (pI) is the specific pH at which the net charge on the molecule is zero (proteins are positively charged at pH values below their pI and negatively changed at pH values above their pI).

A pH gradient is necessary so that under the influence of an electrical field, a protein will move to the position where its net charge is zero. A protein with a positive net charge will migrate towards the cathode becoming progressively less positively charged as it moves through the pH gradient until it reaches a point that corresponds to its pI value.

Isoelectric focusing requires solid support such as agarose gel and polyacrylamide gel. Polyacrylamide is a polymer with small interstices – approximately the size of proteins so that apart from surface charge, separation depends on the size of the molecules. Isoelectric focusing gels contain synthetic buffers called ampholytes that smooth the pH gradients.

Isoelectric focusing needs high voltage (1000 V or more). It gives good separation with a high resolution compared to any other method. Resolution depends on:


 * 1) The pH gradient,
 * 2) The thickness of the gel,
 * 3) Time of electrophoresis,
 * 4) The applied voltage,
 * 5) Diffusion of the protein into the gel.

Method
In the Cyprus laboratory the preferred isoelectric focusing method is the PhastSystemTMwith dry polyacrylamide gels soaked prior to use in a narrow pH gradient (6.7-7.7). The pH gradient is made by Pharmalyte® which generates stable, linear pH gradients in the gels during the run. The haemoglobins migrate under an electric field to a point in the pH gradient that corresponds to their pI (isoelectric point). The separated Hbs on the stained gel are evaluated by visual inspection. Protein patterns from known haemoglobin variants are used as references to identify the protein bands from unknown Hb samples.

Reagents

 * 1) Phast-Gel Dry IEF plates *
 * 2) Pharmalyte pH 6.7-7.7*
 * 3) Kerosene
 * 4) 20% trichloroacetic acid fixative
 * 5) Staining solution stock: one tablet of Phast Gel Blue R tablet * (1 tablet + 80 ml distilled H2O + 120 ml methanol). The solution was stirred for 2 minutes and filtered twice
 * 6) Methanol
 * 7) Destaining solution containing 300 ml methanol + 100 ml acetic acid + 600 ml distilled water (3:1:6).
 * 8) CuSO4 (5H2O)
 * Working solution for staining was made by mixing 30 ml stock staining solution with 270 ml of 3:1:6 destaining solution and 0.45 g CuSO4(5H2O). The solution is filtered and is prepared fresh before use.
 * 1) 0.1% Triton

*These items are provided by the Amersham-Pharmacia company.

Equipment
Pharmacia LKB – Phast System

The system consists of:


 * 1) a separation unit
 * 2) a developing unit for staining procedures.
 * 3) Phast Gel sample – applicator 8/1
 * 4) Phast Gel sample – well stamp
 * 5) Scanning Densitometer LKB Pharmacia

All functions are controlled by a microprocessor.

Gel preparation
Rehydration of dry gels: dry gels are rehydrated using a 1:16 solution of Pharmalyte 6.7-7.7 (100 μl Pharmalyte + 1500 μl distilled H2O). The dry plate is placed with the gel side down on the drop of the solution that has been pipetted on a clean plastic surface, for 1 hr checking at intervals that the plate did not stick to the surface.

After rehydration excess of fluid is removed from the surface of the gel by wiping it gently with the edge of a piece of filter paper.

Sample preparation
Samples are prepared by diluting 5 &mu;l of whole blood in 200 μl 0.1% Triton. The mix is allowed to stand for 5 minutes and then mixed on a vortex mixer. 5 &mu;l is used for the sample application.

Sample application
To load the sample applicator, depressions are formed on a strip of Parafilm (using the PhastGel sample well stamp). 5 &mu;l of each sample is placed on the depressions. The PhastGel sample applicator 8/1 is dipped in the 5 &mu;l droplets on each sample.

Method
 The plates are placed on the cooling bed of the Phast System, on which two drops of kerosene are placed on each plate. After application of the electrodes on the gel the prefocusing stage is performed. After prefocusing, the samples are applied anodally to the gels using the 1 &mu;l applicator. For each type of plate a different separation program on the Phast System is used through the microprocessor of the Phast unit. For running Phast Gel Dry IEF 6.7-7.7 the following separation program is used:  End of separation program.
 * Sample applicator down at 1.2 vh
 * Sample applicator up at 1.3 vh

 The method called separation method 1 was programmed on the Phast System previously and contains three separating steps, Sep 1.1, Sep 1.2 and Sep 1.3. A final step, Sep 1.4, was added to finalise the program. The sample applications will be lowered onto the gels at after 75 vh during Step 1. During this 75 vh period, the sample applicators are loaded. An alarm sounded at 73 vh as a warning that the sample application will occur in 2 vh. The sample applicators are automatically raised at 100 vh of Step 3 and separation proceeds as indicated in the above program.</li> </ol>

Staining
After the focusing steps have been performed, the gels are placed in the staining chamber. The plates are fixed with 20% TCA for 5 minutes at 20 oC, and washed with distilled water for 2 minutes. Subsequently they are stained for 10 min with 0.2% Coomassie Blue (1:10 solution, of stock solution: destain solution), containing 0.15 w/v% CuSO4 (5H2O) at 50 oC.

The plates are destained in the destain solution for 10 min at 50oC and allowed to dry.

Plates are scanned on a laser scanning densitometer for determination of pI values.

Samples containing known haemoglobins (Hb F, Hb S, HbA2, Hb A, Hb Lepore, Hb D) as well as the sample containing the unknown haemoglobin are run on the same plate. The method does not distinguish between Hb G-Philadelphia, Lepore, Hb E and O-Arab.

Interpretation
The identification of the unknown haemoglobin is achieved by measuring the pI value of the unknown haemoglobin in a laser densitometer. The pI values of known haemoglobins, separated on the same plate, are also measured and are used as controls. Despite excellent resolution achieved by isoelectric focusing, accurate quantitation is not possible.