ISSN:
0173-0835
Keywords:
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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 pI 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 (pI〉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 pI, 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 = pK, 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.
Additional Material:
10 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150060202
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