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IEF cards for Amersham WB

IP.com Disclosure Number: IPCOM000248653D
Publication Date: 2016-Dec-22
Document File: 2 page(s) / 133K

Publishing Venue

The IP.com Prior Art Database

Abstract

In isoelectric focusing electrophoresis (IEF), proteins or peptides migrate to their respective isolectric point (pI) in a gel matrix with a pH gradient. IEF is used as the first step in two-dimensional electrophoresis (where the next step is size separation of the focussed proteins in SDS-PAGE). IEF is useful to discriminate small charge differences among protein isoforms. IEF is also used in many different areas: Basic research, Forensics, Pharmaceutical industry, Clinical use, Doping analysis, Food industry, Beverage industry.

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Title: IEF cards for Amersham WB

Introduction

In isoelectric focusing electrophoresis (IEF), proteins or peptides migrate to their respective isolectric point (pI) in a gel matrix with a pH gradient. 

IEF is used as the first step in two-dimensional electrophoresis (where the next step is size separation of the focussed proteins in SDS-PAGE). IEF is useful to discriminate small charge differences among protein isoforms. IEF is also used in many different areas: Basic research, Forensics, Pharmaceutical industry, Clinical use, Doping analysis, Food industry, Beverage industry.

Description

Three basic principles are outlined for IEF applications in the Amerham WB (Western blotting) system:

-       IEF in standard gel cassettes for the Amersham WB system

-       An “IEF card” is used instead of the normal hard plastic part of the gel cassette to provide a possibility to switch polarity in the gel

-       An “IPG DryStrip card” is used in the Amersham WB system to enable IEF in 7 cm precast IPG Strips with different pH gradients

Isoelectric focusing in the Amersham WB system (AWB) is performed with a low density acrylamide gel with Pharmalytes or with Immobilines in a standard gel cassette for the AWB. Running IEF in intact cassettes has two main drawbacks: 1. The sample is loaded on the cathode side, and, 2. Only a part of the gel is scanned in the AWB system.

In most IEF applications, the samples are loaded on the anode (+) side of a pH gradient, except for very basic proteins. Relatively good results may be obtained with cathode loading in intact cassettes with Pharmalytes, but less good results with immobilized gradients.

Therefore an “IEF card” was provided. This has flat electrodes just outside the area that is scanned in the AWB system, and in addition it is wired to switch polarity (see Fig 1). To use the IEF card, the hard plastic part of a gel cassette cast with Pharmalytes or Immobilines is removed and substituted with the IEF card. The IEF card has keying features for precise placement.  

Fig 1. An IEF card for the Amersham WB system.

Samples can be applied anywhere in the area on e.g. paper application pieces or silicon strip wells. The IEF card may for example be provided with 2% Pharmalyte or 7.5% Pharmalyte.

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