2-D gel densitometer for high-contrast and selective imaging of chlorophyll-containing protein complexes separated by non-denaturing polyacrylamide gel electrophoresis

• Published in: Journal of biochemical and biophysical methods Vol. 51; no. 3; pp. 273 - 281
• Main Authors: Ilı́k, Petr, Krchňák, Pavel, Tomek, Pavel, Nauš, Jan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 31.05.2002
• Subjects: CCD densitometry; Chlorophyll - analysis; Chlorophyll a/ b ratio; Electrophoresis, Gel, Two-Dimensional - methods; Electrophoresis, Polyacrylamide Gel - methods; Hordeum - growth & development; Hordeum vulgare; Light-harvesting complex; Light-Harvesting Protein Complexes; PAGE; Photosynthetic Reaction Center Complex Proteins - isolation & purification; Photosystem core; PAGE; Chlorophyll a/ b ratio; CCD densitometry; Photosystem core; Light-harvesting complex
• ISSN: 0165-022X; 1872-857X
• DOI: 10.1016/S0165-022X(02)00029-5

In this work, we present a home-made two-dimensional (2-D) CCD imaging system for the monochromatic densitometry of plane gels and its application to the imaging and densitometry of chlorophyll (Chl)-containing proteins separated by non-denaturing polyacrylamide gel electrophoresis. The monochromatic imaging of separated green bands at the wavelengths corresponding to their absorption band increases their contrast. This allows a better visualization of the faint-green bands in the gel and using of samples with lower Chl content for the electrophoresis. By the comparison of 2-D densitograms of the same gel illuminated with 670 and 650 nm lights, that is, at the red absorption maximum of Chl a and b, respectively, we achieved a selective imaging of the complexes with different Chl a/ b ratio. This approach was used to specify an unknown band that appeared in the gel of the sample prepared from the thylakoid membranes of preheated barley leaves.