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ABSOLUTE QUANTITATION OF SPOTS IN A TWO-DIMENSIONAL DIFFERENTIAL IN GEL ELECTROPHORESIS (2D- DIGE)

IP.com Disclosure Number: IPCOM000249781D
Publication Date: 2017-Apr-04
Document File: 3 page(s) / 56K

Publishing Venue

The IP.com Prior Art Database

Abstract

A technique for absolute quantitation of spots in a two-dimensional differential in gel electrophoresis (2D-DIGE) gel is proposed. The technique includes a lane of protein ladder in the DIGE gel during the second dimension for the DIGE. The protein ladder has proteins with separable molecular weight and placed in increasing amount order. These protein amounts cover the entire dynamic range of the cyanine dyes or florescent dyes.

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ABSOLUTE QUANTITATION OF SPOTS IN A TWO-DIMENSIONAL DIFFERENTIAL IN GEL ELECTROPHORESIS (2D- DIGE)

BACKGROUND

 

The present disclosure relates generally to two-dimensional differential in gel electrophoresis (2D-DIGE) and more particularly to absolute quantitation of spots in a 2D-DIGE gel.

Generally, two-dimensional differential in gel electrophoresis or difference gel electrophoresis (2D-DIGE) analysis allows relative quantitation of spots between samples. There are many instances when estimation of percentage of impurity present in a sample or quantitation of the amount of one specific protein spot present in the protein map of a sample is desired. Minimal labeling involved in conventional 2D-DIGE techniques does not allow such estimations.

One conventional technique uses a single internal standard to quantitate the amount of one protein spot in a sample. However, using single internal standard masks the inherent spots of a sample.

It would be desirable to have an improved technique for absolute quantitation of spots in 2D DIGE).

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 depicts a standard curve plotted for intensity vs. amount and a second-dimension with spot intensities estimated from the curve.

DETAILED DESCRIPTION

A technique for absolute quantitation of spots in a two-dimensional differential in gel electrophoresis (2D-DIGE) gel is proposed. The technique includes a lane of protein ladder in the DIGE gel during the second dimension for the DIGE. The protein ladder has proteins with separable molecular weight and placed in increasing amount order. These protein amounts cover the entire dynamic range of the cyanine dyes or florescent dyes.

According to one embodiment, proteins in the first dimension are pre-labeled in increasing amounts, for example 0.1ng,1ng,10ng,100ng,1000ng and so on with the Cye 3/2/5 dye. The Cye dye is selected based on requirements of the experimental. The pre-labeled proteins are run alongside an immobilized pH gradient (IPG) strip having the DIGE samples in the second dimension. Further, the technique described herein, involves measuring the background subtracted intensities for the bands. For example DeCyder analysis may be used to identify the band as spots to know their intensity and a standard curve of intensity vs. amount is plotted from the same. 2D spot intensities are determined from this curve. The curve can also be modified statistically for better quantitation by increasing the number of data points for the curve by including more standards.

There are many instances when the sample is known and contains fewer spots for example, for bioprocess monoclonal (Mab) soup, and qua...