Densitometric analysis of protein gels

I. Materials and reagents

• Gels stained with colorimetric total protein stain (e.g., Silver Stain, Coomassie Blue)
• chemiSOLO instrument
• chemiSOLO Densitometry Package
• AzureSpot Pro Analysis Software for band quantitation

II. Objective

This note describes a standard procedure for easy and reliable densitometric quantification of proteins separated on 1D and 2D protein gels using the chemiSOLO.

This note includes instructions to:

• verify the calibration (linearity of detection) of the chemiSOLO unit using the 21-Step Calibration Target included with the chemiSOLO Densitometry Package.
• measure optical density (OD) for quantification and identification of proteins separated by gel electrophoresis and stained with colorimetric dyes.

III. Introduction

The identification and quantification of target proteins in complex mixtures is broadly performed through the analysis of images of gels stained with reagents for total protein detection. Among the most-used colorimetric reagents are several formulations for silver staining and many commercial preparations of Coomassie blue dyes.

Different silver stain formulations stain protein spots and bands in a variety of colors including black, blue, brown, yellow, or red, depending on their charge and experimental conditions, which is useful for differentiating overlapping spots in 2D gels.

Some of the advantages of densitometry conducted on the chemiSOLO are:

• High-resolution imaging for small spot identification
• Direct proportionality of the signal over a range of 2.1 OD
• Even transillumination across the imaging area (15 x 10cm)
• Color and grayscale imaging for both qualitative and quantitative analysis
• Verification of the unit calibration

The chemiSOLO offers high resolution imaging to allow for the identification of small bands or spots in 1D or 2D gels. The powerful optics capture signal with direct proportionality using 16-bit data collection to ensure the linearity, sensitivity, and overall accuracy of quantitative analysis.

With an imaging field of view of 15 x 10cm, the chemiSOLO can accommodate most typical gel sizes. The LEDs imbedded in the inner lid provide even transillumination across the imaging stage to ensure consistent sensitivity across the entire field of view.

White light is emitted from the LEDs in the inner lid of the chemiSOLO and passes through the sample to the detector. The absorbance of light by the colorimetric stain in the sample is directly proportional to the concentration of stain bound to the target. Therefore, by detecting light absorbed by the sample, the concentration of stain and the concentration of the target can be detected and quantified.

IV. chemiSOLO verification procedure

Accurate quantification relies on the instrument’s OD calibration. The following procedure describes how to verify the chemiSOLO’s densitometric performance using the 21-Step Tablet included in the chemiSOLO Densitometry Package. The following instructions explain how to acquire an image of the 21-Step Tablet using the chemiSOLO capture software and analyze it using AzureSpot Pro.

For more information about the theoretical basis of densitometry and chemiSOLO verification, please see section VII.

V. Sample analysis procedure

The following procedure describes how to use the chemiSOLO capture software to acquire images of 1D or 2D gels for densitometric analysis.

Sample images may be analyzed using one of the following:

• AzureSpot Pro Analysis Software: 1D gel analysis
• SameSpots 2D Analysis Software: 2D single stain and DIGE analysis
• SpotMap Analysis Software: Host Cell Protein (HCP) Coverage

Analysis of the image using AzureSpot Pro Analysis Software for 1D gel analysis is described below. For 2D gel analysis, follow the instructions in the SameSpots or SpotMap software manuals.

VI. Conclusions

The chemiSOLO was designed for sensitive densitometric quantification of colorimetric gels. With the ability to linearly detect a broad dynamic range of 0 to 2.1 OD, densitometric quantification of visible-stained gels is possible on the chemiSOLO.

VII. Theoretical basis