Abstract

Thermal lens spectroscopy will be developed as a method for ultrasensitive detection and quantitation of proteins separated by gel electrophresis. Low power (15-20 mW) CW lasers will be used to generate thermal lens signals. Configurations providing spatial resolution to 20-50 microns will be developed. Sources of noise in thermal lens spectroscopy of gels will be investigated, and tactics for noise reduction will be devised. Differential configurations for gel or residual stain background absorption subtraction will be designed. Retroreflection will be used to double delivered laser power, to minimize instrument cost. The system will be applied to Coomassie Brilliant Blue and silver-stained systems. Sensitivity increases of approximately 100X over existing instruments are expected. An experimental thermal lens detector for capillary electrophoresis of proteins will be developed. Low energy pulsed laser radiation will be used as the pump light source to provide 190-200 nm excitation. Approximately 100X increase in sensitivity is expected over conventional spectrophotometry. Raman spectroscopy will be used to study the structure of the silver colloids formed by silver staining. The initial protein-silver ion complexes which initiate the reaction will be elucidated by spontaneous Raman spectroscopy. The complexes present in the completed colloids will be studied by surface-enhanced Raman spectroscopy (SERS). SERS will also be employed to measure approximate amino acid content of denatured proteins separated by SDS page and silver-stained.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037006-03
Application #
3291821
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1986-07-01
Project End
1989-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
3
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
University of Michigan Ann Arbor
Department
Type
Schools of Arts and Sciences
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109

  Publications

Rhee, Minsoung; Burns, Mark A (2009) Microfluidic pneumatic logic circuits and digital pneumatic microprocessors for integrated microfluidic systems. Lab Chip 9:3131-43
Al-Hashimi, Hashim M; Walter, Nils G (2008) RNA dynamics: it is about time. Curr Opin Struct Biol 18:321-9
Walter, Nils G; Huang, Cheng-Yen; Manzo, Anthony J et al. (2008) Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat Methods 5:475-89
Rhee, Minsoung; Burns, Mark A (2008) Microfluidic assembly blocks. Lab Chip 8:1365-73
Rhee, Minsoung; Burns, Mark A (2008) Drop mixing in a microchannel for lab-on-a-chip platforms. Langmuir 24:590-601
Walter, Nils G (2007) Ribozyme catalysis revisited: is water involved? Mol Cell 28:923-9
Yamaguchi, Yoshinori; Todorov, Todor I; Morris, Michael D et al. (2004) Distribution of single DNA molecule electrophoretic mobilities in semidilute and dilute hydroxyethylcellulose solutions. Electrophoresis 25:999-1006
Todorov, Todor I; Yamaguchi, Yoshinori; Morris, Michael D (2003) Effect of urea on the polymer buffer solutions used for the electrophoretic separations of nucleic acids. Anal Chem 75:1837-43
Todorov, Todor I; Morris, Michael D (2002) Comparison of RNA, single-stranded DNA and double-stranded DNA behavior during capillary electrophoresis in semidilute polymer solutions. Electrophoresis 23:1033-44
de Carmejane, O; Yamaguchi, Y; Todorov, T I et al. (2001) Three-dimensional observation of electrophoretic migration of dsDNA in semidilute hydroxyethylcellulose solution. Electrophoresis 22:2433-41

Showing the most recent 10 out of 43 publications