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Comparison of three different fluorescent visualization strategies for detecting Escherichia coli ATP synthase subunits after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Author(s) -
Berggren Kiera N.,
Chernokalskaya Elena,
Lopez Mary F.,
Beechem Joseph M.,
Patton Wayne F.
Publication year - 2001
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200101)1:1<54::aid-prot54>3.0.co;2-5
Subject(s) - chemistry , protein subunit , gel electrophoresis , biochemistry , cysteine , atp synthase , mass spectrometry , polyacrylamide gel electrophoresis , time of flight mass spectrometry , chromatography , fluorescence , enzyme , physics , quantum mechanics , gene , ion , organic chemistry , ionization
The correlation between protein molecular weight and the number of lysine or basic amino acid residues was found to be high for broad range molecular weight standards, subunits of Escherichia coli F 1 F 0 ‐ATP synthase and the translated open reading frame of E. coli . A relatively poor correlation between protein molecular weight and the number of cysteine residues was observed in all cases. The ability of amine‐reactive, thiol‐reactive and basic amino acid‐binding fluorophores to detect the eight subunits of F 1 F 0 ‐ATP synthase complex was assessed using 2‐methoxy‐2,4‐diphenyl‐3(2H)‐furanone (MDPF), monobromobimane (MBB) and SYPRO Ruby protein gel stain, respectively. Though experimentally none of the fluorophores provided accurate estimates of the subunit stoichiometry of this complex, MDPF and SYPRO Ruby protein gel stain were capable of semiquantitative detection of every subunit. MBB, however, failed to detect subunits a, b and c of the hydrophobic F 0 complex, as well as subunit ε of the F 1 complex. All three fluorescent detection procedures permitted subsequent identification of representative subunits by peptide mass profiling using matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). The use of thiol‐reactive fluorophores for the global analysis of protein expression profiles does not appear to be advisable as a significant number of proteins have few or no cysteine residues, thus escaping detection.