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Improved and simplified in‐gel sample application using reswelling of dry immobilized pH gradients
Author(s) -
Sanchez JeanCharles,
Rouge Véronique,
Pisteur Michel,
Ravier Florence,
Tonella Luisa,
Moosmayer Marlis,
Wilkins Marc R.,
Hochstrasser Denis F.
Publication year - 1997
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.1150180305
Subject(s) - chromatography , immobilized ph gradient , reagent , precipitation , sample preparation , chemistry , materials science , analytical chemistry (journal) , isoelectric focusing , organic chemistry , physics , meteorology , enzyme
A simple and inexpensive methacrylate rehydration chamber was built to accomodate ten immobilized pH gradient (IPG) strips. In the chamber, entire IPG gels were used for sample application, with the protein entering the gels during their rehydration. For rehydration, commercially available or laboratory‐made strips were positioned in the grooves with the gel in contact with 500 μL of sample for 6 h or overnight. This avoided the use of sample cups, eliminated precipitation at the sample application site, thus improving resolution over the entire pH range of the gels. It also allowed precise control of protein amounts and sample volumes loaded into the IPG gels, and also lowered costs of reagents during rehydration and equilibration owing to the reduced volumes. Up to 5 mg of protein can be loaded on wide IPG gels and up to 15 mg of some samples on narrow pH range gels.