Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels

Pier Giorgio Righetti, Cecilia Gelfi

Research output: Contribution to journalArticle

Abstract

A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135-172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40-45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to ba carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37°C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.

Original languageEnglish
Pages (from-to)103-119
Number of pages17
JournalJournal of Biochemical and Biophysical Methods
Volume9
Issue number2
DOIs
Publication statusPublished - 1984

Fingerprint

Proton-Motive Force
Isoelectric Focusing
Gels
Proteins
Immobilized Proteins
Load limits
Fractionation
Charge density
Sepharose
Chemical properties
Polymers
Casting
Recovery

Keywords

  • immobilized pH gradients
  • isoelectric focusing
  • preparative electrophoresis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

Cite this

Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels. / Righetti, Pier Giorgio; Gelfi, Cecilia.

In: Journal of Biochemical and Biophysical Methods, Vol. 9, No. 2, 1984, p. 103-119.

Research output: Contribution to journalArticle

@article{0bf40db2538e43a59da9d44efbe57d8d,
title = "Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels",
abstract = "A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135-172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5{\%}T gels an upper load limit of 40-45 mg protein/ml gel volume is found, in 2.5{\%}T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to ba carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a {\%}T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37°C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5{\%}T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.",
keywords = "immobilized pH gradients, isoelectric focusing, preparative electrophoresis",
author = "Righetti, {Pier Giorgio} and Cecilia Gelfi",
year = "1984",
doi = "10.1016/0165-022X(84)90002-2",
language = "English",
volume = "9",
pages = "103--119",
journal = "Journal of Biochemical and Biophysical Methods",
issn = "0165-022X",
publisher = "Elsevier BV",
number = "2",

}

TY - JOUR

T1 - Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels

AU - Righetti, Pier Giorgio

AU - Gelfi, Cecilia

PY - 1984

Y1 - 1984

N2 - A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135-172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40-45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to ba carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37°C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.

AB - A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135-172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40-45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to ba carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37°C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.

KW - immobilized pH gradients

KW - isoelectric focusing

KW - preparative electrophoresis

UR - http://www.scopus.com/inward/record.url?scp=0021433331&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021433331&partnerID=8YFLogxK

U2 - 10.1016/0165-022X(84)90002-2

DO - 10.1016/0165-022X(84)90002-2

M3 - Article

C2 - 6736557

AN - SCOPUS:0021433331

VL - 9

SP - 103

EP - 119

JO - Journal of Biochemical and Biophysical Methods

JF - Journal of Biochemical and Biophysical Methods

SN - 0165-022X

IS - 2

ER -