Analytical Biochemistry 1996-06-15

Ultramicro-analysis by use of light-scanning photoacoustic densitometry for electrophoresed protein in human hair.

T Fukami, K Uchiyama, Y Yoshimura, T Watanabe, H Nakazawa

Index: Anal. Biochem. 238(1) , 60-4, (1996)

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Abstract

A technique was developed for the ultramicro-analysis of proteins electrophoresed by Laemmli's method using a light-scanning photoacoustic densitometer. After electrophoresis, the proteins were blotted on a nitrocellulose membrane filter and colored by the avidin-biotin complex method. This filter was then measured using a photoacoustic densitometer. The optimal blotting time was 150 min. The relative standard deviation of four measurements was 3.89% for 200 ng bovine serum albumin (BSA). The limits of detection were 2.3, 0.69, 4.4, and 2.9 ng (S/N = 3) for BSA, ovalbumin, carbonic anhydrase, and alpha-Lactoalbum respectively. Proteins eluted from hair by various harmful agents, such as a surfactant and UV irradiation, were analyzed by the present method. Using 2-mercaptoethanol, the molecular weights of proteins in hair were in the range of 14,000-64,000. Maximal elution of protein was obtained at pH 8. More protein was eluted under alkaline conditions than under acidic conditions. A protein of Mr 68,000 was eluted from hair by oxidative treatment with either UV irradiation or sodium bromate.