The methodological work of the year served the purpose of adaptation of originally macromolecular techniques to the separations of subcellular particles and membrane complexes. Theoretical interpretation of protein electrophoresis data led to the important insight in that regard that zone dispersion, a key element of resolving power, related to interactions with either gel fibers or walls of the vessel at capillary distances. That insight guided the development of a preparative method for large particles. Resolving power of separations was also augmented by the finding that power supply interrupted by rest periods allowing for Joule heat dissipation yielded at least a doubling of field strength, and surprisingly, that a derivatized, highly soluble agarose yielded a 4 to 5 fold increase in resolution over the conventional medium for protein separations, polyacrylamide. Automated instrumentation originally designed for gel electrophoresis had been shown last year to be applicable to large particles, replacing gels by concentrated polymer solutions. This technique has now been transformed into a preparative one for subcellular-sized particles. The wide applicability of the automated gel electrophoresis technique has also been advanced during the previous year by demonstration of its applicability to native, non-fluorescently labeled, particles. The originally poor detection sensitivity of that method has now been greatly improved by use of the fluorescence enhancer, salicylate, to control and maximize the level of the baseline for detection by """"""""fluorescence reduction"""""""".

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Intramural Research (Z01)
Project #
1Z01HD000171-20
Application #
2575606
Study Section
Special Emphasis Panel (LTPB)
Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
1996
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Cretich, Marina; Stastna, Miroslava; Chrambach, Andreas et al. (2002) Decreased protein peak asymmetry and width due to static capillary coating with hydrophilic derivatives of polydimethylacrylamide. Electrophoresis 23:2274-8
Chrambach, Andreas; Griffith, Ann L (2002) Reflections on the first anniversary of N. Catsimpoolas' death. J Biochem Biophys Methods 52:1-10
Radko, Sergey P; Chang, Huan-Tsung; Zakharov, Sergey F et al. (2002) Electroelution without gel sectioning of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis: fluorescent detection, recovery, isoelectric focusing and matrix assisted laser desorption/ionization-time of flight of the electroeluate. Electrophoresis 23:985-92
Yefimov, S; Sjomeling, C; Yergey, A L et al. (2001) Stacking of unlabeled sodium dodecyl sulfate-proteins within a fluorimetrically detected moving boundary, electroelution and mass spectrometric identification. Electrophoresis 22:999-1003
Chang, H T; Yergey, A L; Chrambach, A (2001) Electroelution of proteins from bands in gel electrophoresis without gel sectioning for the purpose of protein transfer into mass spectrometry: elements of a new procedure. Electrophoresis 22:394-8
Yefimov, S; Yergey, A L; Chrambach, A (2001) Sequential electroelution and mass spectroscopic identification of intact sodium dodecyl sulfate-proteins labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester. Electrophoresis 22:2881-7
Buzas, Z; Chang, H T; Vieira, N E et al. (2001) Direct vertical electroelution of protein from a PhastSystem band for mass spectrometric identification at the level of a few picomoles. Proteomics 1:691-8
Li, Y M; Chrambach, A (2001) Gel electrophoretic isolation, in the hundred microgram range, of recombinant SDS-syntaxin from sea urchin egg cortical vesicles. Prep Biochem Biotechnol 31:369-87
Chiari, M; Cretich, M; Stastna, M et al. (2001) Rapid capillary coating by epoxy-poly-(dimethylacrylamide): performance in capillary zone electrophoresis of protein and polystyrene carboxylate. Electrophoresis 22:656-9
Buzas, Z; Li, T; Chrambach, A (2001) Horizontal gel electrophoresis of SDS-proteins on the PhastSystem with an at least 25-fold increased protein load volume. Anal Biochem 292:161-3

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