Health Information

Gel preparation of Isoelectric focusing gel electrophoresis

The carrier ampholytes, riboflavin and acrylamide solution with covering suitable pH range mixture is poured over a glass plate (25×10cm) that contains a spacer. The 2nd glass plate is then placed on top of the first to form gel cassette. Gel is polymerized by photo polymerization by placing the gel in bright light for 2-3 hrs. Once the gel is set potential difference is applied when ampholytes form a pH gradient between anode and cathode. 4% polyacrylamide gels are commonly used; agarose can also be used specially for the study of high molecular weight protein. That may undergo some sieving at low % acrylamide gel.

Filter paper wicks used in Isoelectric focusing gel electrophoresis

Wicks 3mm thick are used. These wicks are wetted in H3PO4 and NaOH are placed on the anode and cathode ends of plate. The wicks are inturn connected to protein wire electrode.

Power pack

To achieve rapid separation high voltage of 2500V are used. Considerable heat is produced hence gels are run on cooling plates 10°c. Power packs with stabilizers that stabilize the power out put to minimize thermal fluctuation are used.

Application of Isoelectric focusing gel electrophoresis

To determine the Isoelectric point of unknown protein

Isoelectric protein of a particular protein can be determined by running a mixture of protein of known isoelectric point on the same gel. After staining the distance of each band from one electrode is measured and a graph of distance for each protein against its isoelectric point is plotted. Using this calibration line, isoelectric point of an unknown protein can be determined from its position on the gel.

Used for studying micro heterogeneity of protein

Isoelectric focusing is a highly sensitive analytical technique and is useful for studying microheterogeneity in a protein.
For example, a protein may show a single band on a SDS added but show 3 bands on an Isoelectric focusing gel. This may occur when protein exits in mono, di and tri phosphorylated form.
The difference of number of a phosphate group has no significant effect on the overall relative molecular mass of protein. Hence a single band on SDS- added gel, but small charge difference introduces on each molecule can be detected by Isoelectric focusing.

Used for separating isoenzymes

Isoenzymes are different forms of the same enzyme often differing by only one or two amino acid residue. This approach is found useful in forensic science. It is used for analytical separation; it can also be used for preparative purpose.