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Proteomic ‘contigs’ of Ochrobactrum anthropi , application of extensive pH gradients
Author(s) -
Wasinger Valerie C.,
Bjellqvist Bengt,
HumpherySmith Ian
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150180812
Subject(s) - proteome , immobilized ph gradient , isoelectric focusing , contig , isoelectric point , electrophoresis , chemistry , two dimensional gel electrophoresis , proteomics , chromatography , protein purification , gel electrophoresis , computational biology , biology , biochemistry , genome , gene , enzyme
The most extensive linear pH gradients yet employed in combination with two‐dimensional gel electrophoresis are described, along with their application in proteome analysis. A significant proportion of the protein compliment of bacterial species is believed to be accessible using an extended linear pH gradient of 2.3 to 11.0. Protein standards with predicted isoelectric points (p I ) ranging from 3.24 to 9.56 were used to confirm focusing positions with respect to the immobilised pH gradients (IPG) prior to mapping studies of Ochrobactrum anthropi . Multiple gel images were used to construct contiguous windows of protein expression (‘proteomic contigs’) within 18 cm pH gradients 2.3–5, 4–7, and 6–11 in conjunction with 15%T and 7.5%T acrylamide gels, the latter being used to resolve higher molecular weight ( M r ) proteins. Each IPG had a 5 cm region of similar pH gradient overlap at pH 4–5 and pH 6–7 that was used to construct an image of protein expression characteristic of whole cell lysates. This is reminiscent of genomic sequencing initiatives whereby portions are combined to form a contiguous picture of the whole. The protein maps obtained demonstrated a means of resolving the many tens of thousands of cellular proteins likely to occur in eukarvotic systems, but also highlighted the need to further optimise protein extraction, equilibration buffers, and separation conditions of higher M r proteins occurring at extreme p I . Theoretical 2‐D protein maps were constructed for five organisms for which the total DNA sequence is now available. In all cases, higher M r acidic and basic proteins were shown to be common.