Two-dimensional maps in soft immobilized pH gradient gels: A new approach to the proteome of the Third Millennium

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Abstract
Same major improvements in proteome analysis of cytosolic and membrane proteins by two‐dimensional mapping are here reported. A much improved transfer of proteins from the first to the second dimensional sodium dodecyl sulfate (SDS)‐gel is obtained by simply diluting the gel matrix, normally composed of 4%T polyacrylamide in all commercially available Immobiline strips down to as low as 3%T. In the analysis of total lysates of platelets, this augmented transfer has been evaluated as being 2–3 times higher than in standard 4%T gels. A second major improvement, in the case of analysis of membrane protein preparations, has been demonstrated to consist in a delipidation step in a tertiary solvent mixture composed of tri‐n‐butyl phosphate:acetone:methanol in a 1:12:1 ratio. By adopting this protocol, large amounts of spectrins (240–220 kDa, filamentous proteins of the red blood cell membranes) could be transferred vs. essentially none when delipidation was omitted. The present report also confirms the importance of a reduction and alkylation step of the protein sample prior to all electrophoretic steps, including focusing in the Immobiline gel, as recently reported by Herbert et al. (Electrophoresis 2001, 22, 2046–2057).