Electrophoresis and Sample Preparation for SRXRF Measurement

Flatbed gel electrophoresis (GE) is used extensively in the analysis and characterization of proteins with a variety of protocols such as isoelectric focusing (lEF) and polyacrylamide gel electrophoresis (PAGE), but has not been specifically developed for the separation of metal-containing proteins. Therefore, before using the method for speciation of biological samples, some important parameters have to be checked. It has to be investigated in detail if speciation data are hampered by contamination or alteration of native species due to the influence of the electric field and/or denaturing buffer systems.  

Following the separation, proteins are generally visualized by staining the gel with one of a range of stains (typically silver or Coomassie blue). It has been demonstrated that staining the gel using silver or Coomassie blue can result in loss of most metals from their bound proteins. To avoid the loss of metals from protein bands and the contamination of sample resulting from the use of colorant and decolorant, two electrophoresis gel must be loaded with the same sample and run simultaneously, the first is used for XRF analysis and the second is colored and used for protein quantification. The inability to stain the gel before SRXRF measurement requires the use of scan mode for XRF 

Hydrogenase enzyme HiPIP Detection of Fe, Ni 3 MeV proton beam SDS-PAGE 31-33 

Mixture of 6 proteins Quantification of Fe, Cu, Zn 2.5 MeV proton beam SDS-PAGE 34,35 

Photosystems PSI RC in thermo- Quantification of Fe 2 MeV proton beam SDS-PAGE 36 

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