XRF Methods Used for Elemental Analysis in Protein Fractions after Biochemical Separation

1 XRF Methods Used for Elemental Analysis in Protein Fractions after Biochemical Separation 

TXRF has been used to quantify metals in protein bands in embryogenic callus (Citrus sinensis L. Osbeck) after separation by SDS-PAGE. The metal-contaiiung bands were detected by micro-SRXRF. The gel was then decomposed in a microwave oven and the metal-binding protein was quantified by synchrotron radiation total reflection X-ray fluorescence (SR-TXRF). Signals that were almost free of background noise could be obtained.  

In spite of the great efforts made to develop TXRF as a promising technique, its widespread implementation in speciation analysis, combined with HPLC, has been hampered by the lack of automation for (1) depositing the eluted fractions onto TXRF carriers, (2) the possible repeated collection for preconcentration purposes, (3) drying, and (4) element analysis. Such automation would speed up the investigations significantly.  

In order to investigate the suitability of a combination of SDS-PAGE and SRXRF in the detection of metal-containing proteins, Weseloh et al. have chosen metal-loaded apoazurin and selenoproteins in rat testis homogenate as examples for non-covalently bound metals and for covalently bound trace element, respectively, to obtain some basic information. After SDS-PAGE separation, the proteins blotted onto a polyvinylidene difluoride (PVDF) membrane and then used for SRXRF measurements at the beamline L at HASYLAB. The pilot study showed that the combination of protein separation 

Samples beside being shaken in water also dialysed in water for 42 h 

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