Isofocusing Chromatography Prepared by- Shirin Akhter

Introduction Iso-focusing chromatography is a chromatography technique that separates protein according to differences in their isoelectric point (PI). Its a polishing step for partially purified samples. Its a high resolution technique and can be used for analytical separation. We can be used this technique as a complementary technique when the protein is not resolved by other chromatography technique such as ion exchange, size.

Relationship between protein PH and net charge Overall net charge of the protein changes according to the PH. The PI of each protein is the PH at which the protein has zero surface charge. When the PH above its isoelectric point, protein will bind to positively charged medium or anion exchanger. When the PH below its PI, protein will bind to negatively charged medium or cation exchanger. In isofocusing technique, proteins with different PIs can be separated by being passed through the chromatofocusing column which is packed with a specifically designed medium. Here the PH gradient is generated on the column by specifically designed and matched amphoteric buffers. Proteins elute in order to their isoelectric point.

Figure- Separating proteins according to their isoelectric points by chromatofocusing.

Mechanism of separation Protein separation according to their different PIs, the chromatofocusing medium is equilibrated with start buffer at high PH. An elution buffer in the column begins to titrate the amines on the medium and the proteins. After pre-gradient volume of elution buffer has passed, sample is applied to the column. Proteins in the sample that are at PH above their PI are negatively charged which retained near top of the column.

Proteins that are at a PH below their PI begin to migrate down the column and will not bind until their PH above their PI. When the proteins migrate down the column, PH of surroundings increases and proteins reaches at PH above their PI which will bind column again. The protein with highest PI elute first and the protein with lowest PI elute last.

Figure: Proteins with different PIs separates as they pass through the column.

Media and buffer for this technique We can use different types of media for this technique such as- Mono P- for fast separation. Mono P 5/200 GL- for highest resolution separation. PBE 94- for scaling up from Mono P. PBE 118- when PH gradient above PH 9.

Different buffers based on the PH gradient- Polybuffer 74- for PH gradient between 7 and 4. Polybuffer 96- for PH gradient should begin above PH 7. Pharmalyte- PH

Drawback for this technique It is less suitable for the isolation of proteins that precipitate irreversibly at or near their isoelectric point because these proteins are likely to precipitate on the column if they reach a high enough concentration.

Isoelectric focusing: This is also a high resolution method for the separation of proteins, peptide and enzymes. Principle of this method: In a pH gradient the sample components migrate towards the anode or the cathode to the pH values, where their net charges are zero: their isoelectric points (pI).

Focusing effect: A protein diffuse away from its pI, it would gain a charge and migrate back. Estimation of isoelectric point for the protein: The isoelectric points of the proteins can be estimated with a calibration curve using marker proteins.

Equipment: Isoelectric focusing (IEF) is mostly run in horizontal gels. The reason behind to run this method in horizontal gels: As IEF separates only according to the charge, the gel matrix must contain large pore sizes. Such a soft gel can slide down between vertical glass plates. The gel should preferably be cast on a film support. Isoelectric focusing requires efficient cooling and exact temperature control. This is optimally achieved on a horizontal ceramics cooling plate connected to a thermostatic circulator, or a peltier cooling plate.

Samples have to be applied on a defined pH location within the pH gradient in order to avoid aggregation and precipitation of some proteins. This is only possible on a horizontal gel with an open surface. Sharply focused bands can only be obtained with a high field strength, high voltages have to be applied. Only horizontal equipment can meet the necessary safety precautions.

Sample application: The samples are applied on the surface of the gels. The mode of sample application is dependent on the sample composition and the IEF gel type. Because the gradient is sensitive to high salt and buffer concentrations, it is very helpful to desalt the samples before application.

The procedure of an IEF run: The running condition should be optimized because it is very important to prevent precipitation and aggregation of proteins, and to achieve good reproducibility. Temperature control is important, because pIs are highly dependent on the temperature. Prefocusing phase should be performed in order to establish the gradient. Samples have to be loaded on the optimized location with the optimized mode.

Sample entry should be performed at low field strength to prevent aggregation. The separation time is a compromise between letting all proteins reach their pIs and keeping the gradient drift to a minimum. After the run, the proteins are fixed by immunofixation and stained for visualization.

Conclusion: Although isofocusing is foreign to many process developers, their fractionation characteristic has advantage to serious evaluation on a routine basis. It has limitations and it has a distinct set of development variables, but there are neither more limitations nor variables than any other technique. They are simply different. For bulk separation, it is just as powerful as salt-gradient ion exchange, hydrophobic interaction, or size exclusion chromatography.

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