Spectrophotometric Quantitation of Silver in X-Ray Film Images and Silver-Stained Electrophoresis Gel Bands

• Published in: Analytical biochemistry 1995, Vol.224 (1), p.190-194
• Main Authors: Sanchez, R.I., Darsley, M.J., Martin, M.T.
• Format: Article
• Language: English
• Subjects: Electrophoresis, Polyacrylamide Gel; Silver - analysis; Silver Staining; Spectrophotometry; X-Ray Film
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1995.1028

This paper describes a novel spectrophotometric method for quantitating the silver in silver-stained electrophoresis gel bands and X-ray film images. Silver in excised areas is quantitated by (i) oxidation/solubilization of Ag 0 to Ag + with nitric acid, (ii) neutralization followed by extraction into an organic solution of dithizone (diphenylthiocarbazone), and (iii) visible absorbance measurement at 600 nm. Spectrophotometric quantitation is possible because dithizone undergoes a dramatic color change from blue-green to yellow upon binding Ag + (Δ ϵ 600 = 3.28 × 10 4 M −1 cm −1). Experiments with silver-stained gels and with X-ray film images obtained by chemiluminescence and autoradiography demonstrated that the color change relates to the amount of silver present and hence to the amount of material represented by the band or image. This method is a convenient, inexpensive alternative to the use of a densitometer to quantitate silver-containing images.