Preparative isoelectric focusing purification

KHALYFA A, KERMASHA S, KHAMESSAN A, MARSOTP and ALLI I. 1993. Purification and characterisation of chlorophyllase from alga (Phaeodactylum tricornutum) by preparative isoelectric focusing. Biosci Biotech Biochem 57(3) 433-437. 

Recommendation Employ where (1) the other assays show interference, especially with complex sample buffers used in one-dimensionaP and two-dimensional electrophoresis (2) the amount of sample is limited and/or reprobing of the dotted samples is desirable or (3) the detection of protein in aliquots from column chromatography or preparative isoelectric focusing on protein purification is needed (spot 2 fi onto membrane, process according to standard protocol, view destained membrane sheets under UV illumination). 

Preparative isoelectric focusing systems in free solutions like recycling isoelectric focusing in rotating tubes [8] and multicompartment electrolysers employing isoelectric membranes [9] have been developed for prefractionation and purification of proteins from highly heterogeneous mixtures. 

Fully equipped protein purification laboratories should also have preparative electrophoresis and isoelectric focusing apparatuses for rare occasions when other techniques fail to give sufficient separation. 

Isoelectric focusing is a mature separation technique that has a place in any laboratory doing work with proteins. The analysis of a protein is not complete without a determination of its isoelectric point and all protein databases have at least estimates of the pis of the represented proteins. Proteins thought to be pure by other methods are often found to be mixtures of several proteins when analyzed by IEF. Isomeric forms of the same protein that are revealed by IEF are valuable indicators of mutations or differences in posttranslational modifications. IEF plays a crucial role in 2-D PAGE and preparative IEF allows for high-purity fractionations of unparalleled resolution. A simple keyword search of literature databases shows that about 500 journal articles are written per year referring to IEF. This amply attests to the value of IEF as a tool for protein analysis and purification. 

Cellobiose or cellulose were necessary to induce the production of fS-D-glucosidase activity by Sporotrichum pulverulentum. Purification via preparative slab gel isoelectric focusing and affinity chromatography on a phenyl derivative of agarose gave five active fractions (p/ s 4.5—5.2, mol. wts. 1.65—1.82 X 10 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). 

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