Thermal lens spectrometry applied for a sensitive detection of silver-stained protein bands in polyacrylamide gels

Abstract

Proteins separated by gel electrophoresis are commonly quantified by colorimetric analysis of the protein band stain intensity. For the most sensitive stain (silver), the minimal protein amount, needed for the optical visualization of the band, is around 1ng. This study investigates the use of thermal lens (TL) spectrometry for the quantification of subnanogram protein amounts. The developed dual laser TL sensor is set up in collinear geometry; it includes a HeNe probe beam, a 450nm diode laser pump beam, and a gel holder. After silver staining, the polyacrylamide gel is scanned by the sensor while kept soaked in water, and in 50% (v/v) methanol/water. During scanning, the TL signal of each band is recorded in function of its position. The TL sensor can detect protein amounts as low as 0.1ng in water, and 0.05ng in 50% methanol/water. The limit of detection ranges from 8 to 50pg, depending on the soaking medium and the staining strength. The theoretical results, predicting the sensitivity enhancement in methanol/water, are in agreement with the results. In conclusion, thermal lens spectrometry proves to be a valid method for the detection of subnanogram protein amounts in polyacrylamide gels, and its application can be extended to other soft gel matrices.