CHROMATOFOCUSING

• Chromatofocusing is a form of gradient elution chromatography performed using ion exchange resin column and an internally developed pH gradient that travels through the column as the retained front

• This technique was developed by sluyterman and his colleagues

• Variant of ion exchange chromatography- fast-high capacity-high resolution-unique selectivity-powerful column chromatography

• This is usually used for final polishing of proteins

• This is exclusively for proteins

• This technique is all about separation of proteins according to their isoelectric point (PI)

• This technique is a bit similar to isoelectric focusing but here no electric field is involved instead a pH gradient is made to propagate inside an ion exchange chromatography

• Separation between protein isoforms differing by a single amino acid residue and by less than 0.05 pH unit in apparent isoelectric point

• The pI of each protein is the pH at which the protein has zero surface charge.

• Proteins with different pIs can be separated by being passed through a chromatofocusing column

procedure

• the chromatofocusing medium is equilibrated with start buffer at a pH slightly above the highest pH required.

• The elution buffer(polybuffer) is passed through the column and begins to titrate the amines on the medium and the proteins.

• Thus a gradient pH is developed

• sample is applied to the column by mixing it with the start buffer

• Proteins in the sample that are at a pH above their pI are negatively-charged and retained near the top of the column

• proteins that are at a pH below their pIbegin to migrate down the column and bind as they reach the zone where the pH is above their pI

•The protein with highest PI elute first and the protein with lowest PI elute last.

PACKING OF COLOUMN

• Column is packed with monobeadmatrix

• Degas the start buffer and the slurry to avoid air bubbles which can interfere within the separation

BUFFERS FOR WIDE pH INTERVAL

BUFFERS FOR NARROW pH RANGE

LIMITATION

• 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.

Presented by,

Monisha jayabalan