Identification of Phosphoamino Acids Paper Electrophoresis

Amino acid paper electrophoresis has sufficient sensitivity and reproducibihty for amino acid analysis. If analysis by HPLC is not available in or around your lab, paper electrophoresis may be an alternative. 

A glacial acetic acid/pyridine/ddH20 50 5 945 (v/v/v), pH 3.4 B ninhydrin spray 0.3 g ninhydrin dissolved in 100 ml n-butanol 

Electrophoresis is done on ready-to-use cellulose plates or on chromatography paper (e.g., Whatman No. 3MM, Schleicher and Schuell No. 2040b, Macherey and Nagel MN) of 20 cm length. 

The protein or peptide sample is hydrolyzed in a closed tube at 110 °C for 2h . After hydrolysis the samples are lyophilized, dissolved in a small volume of ddH20 and lyophilized again. 

The start area on the plate (strip) is marked with a pencil at the cathodic side. If several samples should be analyzed in the same run, the start areas should have a distance of 1.5-2 cm. A plastic sheet covers the starting zone and the plate is sprayed with Soln. A until it is humid but not wet. 

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