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Preparative two‐dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

Preparative two‐dimensional gel electrophoresis with agarose gels in the first dimension for high... A two‐dimensional gel electrophoresis (2‐DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2‐DE) was compared with an immobilized pH gradient 2‐DE method (IPG‐Dalt). The former method was shown to produce significant improvements in the 2‐D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2‐DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2‐mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA‐free), 1% Triton X‐100 and 3% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second‐dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2‐DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first‐dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5—4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2‐DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Electrophoresis Wiley

Preparative two‐dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

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References (18)

Publisher
Wiley
Copyright
"Copyright © 2000 Wiley Subscription Services, Inc., A Wiley Company"
ISSN
0173-0835
eISSN
1522-2683
DOI
10.1002/(SICI)1522-2683(20000501)21:9<1653::AID-ELPS1653>3.0.CO;2-9
pmid
10870952
Publisher site
See Article on Publisher Site

Abstract

A two‐dimensional gel electrophoresis (2‐DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2‐DE) was compared with an immobilized pH gradient 2‐DE method (IPG‐Dalt). The former method was shown to produce significant improvements in the 2‐D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2‐DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2‐mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA‐free), 1% Triton X‐100 and 3% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second‐dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2‐DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first‐dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5—4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2‐DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.

Journal

Electrophoresis – Wiley

Published: Jan 1, 2000

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