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Preparative isoelectric focusing in immobilized pH gradients IV. Recovery of proteins from Immobiline matrices into ion‐exchange resins
Author(s) -
Casero Patrizia,
Gelfi Cecilia,
Righetti Pier Giorgio
Publication year - 1985
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150060202
Subject(s) - chemistry , chromatography , elution , electrophoresis , agarose , isoelectric point , tris , buffer (optical fiber) , ion exchange , titration , isoelectric focusing , buffer solution , immobilized ph gradient , formate , ion , inorganic chemistry , enzyme , biochemistry , organic chemistry , telecommunications , computer science , catalysis
A new electrophoretic transfer system for the recovery of proteins focused in Immobiline matrices is described. The gel strip containing the sample zone of interest is transferred to a horizontal tray and embedded in 1%, low‐gelling (37°C) agarose. For acidic to neutral proteins (up to p I 7.7) the electrophoretic transfer is from the IPG strip into a layer of DEAE‐Sephadex, buffered at pH 8.5 in 100 mM Tris‐acetate. Recovery (better than 90% in all cases studied) is achieved by titrating the resin at pH 9.5, in 200 mM Tris‐Gly buffer, containing 200 mM salt. For basic proteins (p I >7.7) the electrophoretic retrieval is from the IPG strip into a zone of CM‐Sephadex, buffered at pH 6.0, in 50 mM citrate (cathodic migration). Recovery (again better than 90%) is accomplished by titrating the exchanger at pH 4.0, in 200 mM formate buffer, containing 200 mM NaCl. Exposure to pH 9.5 does not affect enzyme activity if the eluate is promptly titrated around neutrality; a pH 4 milieu might irreversibly alter enzymes, in which case elution is best performed by titrating the protein to its p I , rather than protonating the exchanger. It has been demonstrated that Immobiline gels, even when incorporating 5 times the standard amount of buffer (75 mM Immobiline at pH = p K , i. e. 50 mM buffering ion and 25 mM titrant) exhibit, under the electric field, negligible ion‐exchange properties, thus ensuring ideal behavior as supports for isoelectric focusing.