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Sumner Group: Silver Destaining Using Hydrogen Peroxide

Protocol

Step Repeats Time Solution Purpose
1 2X 5 min Water Remove acid and PAGE buffers
2 2X 5 min 25mM NH4HCO3 pH adjustment and silver complexation

3

 1X Until complete (<10 min) 1% H2O2 in 25mM NH4HCO3 Silver oxidation and removal
4 2X 5 min Water H2O2 and Ag+ removal
5 1X 5 min 1% Formic acid Lower pH of gel plug
6 1X 5 min 50% Water: 50% acetonitrile w/1% Formic acid Sequential dehydration of gel band/plug
7 1X 10 min Acetonitrile Dehydration of gel band/plug

Recommended volumes are 25µL for 1mm diameter gel plugs and 50 µL for bands: however, these can be adjusted to meet need or personal preference

Reference:
[1] - Rapid Commun. Mass Spectrom. 2002; 16: 160-168

Shen, G., Huhman, D. V., Lei, Z., Snyder, J., Sumner, L. W., & Dixon, R. A. (2012). Characterization of an isoflavonoid-specific prenyltransferase from Lupinus albus. Plant Physiology, March, doi:10.1104/pp.112.195271.
Lei, Z., Li, H., Chang, J., Zhao, P. X., & Sumner, L. W. (2012). MET-IDEA version 2.06; improved efficiency and additional functions for mass spectrometry-based metabolomics data processing. Metabolomics, February, doi:10.1007/s11306-012-0397-5.
Tzin, V., Malitsky, S., Moyal Ben Zvi, M., Bedair, M., Sumner, L. W., Aharoni, A., & Galili, G. (2012). Expression of a bacterial feedback-insensitive 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase of the shikimate pathway in Arabidopsis elucidates potential metabolic bottlenecks between primary and secondary metabolism. New Phytologist, February, doi: 10.1111/j.1469-8137.2012.04052.x.
Ruiz-May, E., De-la-Pena, C., Galaz-Ávalos, R. M, Lei, Z., Watson, B. S., Sumner, L. W., & Loyola-Vargas, V. M. (2011). Methyl jasmonate induces ATP biosynthesis deficiency and accumulation of proteins related to secondary metabolism in Catharanthus roseus (L.) G. hairy roots. Plant Cell & Physiology, , doi:10.1093/pcp/pcr086.
Tadege, M., Lin, H., Bedair, M., Berbel, A., Wen, J. Q., Rojas, C. M. , Niu, L., Tang, Y., Sumner, L. W., Ratet, P., McHale, N. A. , Madueno, F, & Mysore, K. S. (2011). STENOFOLIA regulates blade outgrowth and leaf vascular patterning in Medicago truncatula and Nicotiana sylvestris. Plant Cell, 23 (6), 2125-2142 doi:10.1105/tpc.111.085340.
Lei, Z., Huhman, D. V., & Sumner, L. W. (2011). Mass spectrometry strategies in metabolomics. Journal of Biological Chemistry, 286 (29), 25435-42 doi:10.1074/jbc.R111.238691.
Current External Research Support
  • The Consortium for Legume Research: An integrated approach to cotton root rot disease of alfalfa
  • Functional Analysis of Pollen Exine Assembly
  • GEPR: Comparative genomics of secretory trichomes - biofactories for production of plant secondary metabolites
  • An Integrated Functional Genomics Approach towards the Understanding of Artemisinin Metabolism
Current Internal Research Support
  • Spatially Resolved and Integrated Transcriptomics, Proteomics, and Metabolomics of Medicago sativa Border Cells
Prior External Research Support
  • An integrated approach to functional genomics and bioinformatics in a model legume
  • Metabolomics: A functional genomic tool for deciphering functions of Arabidopsis genes

Medicago Handbook

  • Pedro Mendes
    Virginia Bioinformatics Institute
    Blacksburg, VA
  • Tim Smith
    Chemistry, Computer and Physical Science
    Southeastern Oklahoma State University
    Durant, OK
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