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Wide bore tube electrophoresis using Pluronic polymer gels in conjunction with spectrophotometry, HPLC, and MALDI/MS

Wei, Wenjun
http://rave.ohiolink.edu/etdc/view?acc_num=miami1375056442

Abstract Details

2013, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
This dissertation develops a novel electrophoresis approach to analyze the often-lost low molar mass peptides and proteins (<8 kDa), not amenable for separation by polyacrylamide gel electrophoresis. This method uses a wide bore glass tube (1 mm I.D. x7 cm length) filled with thermal-responsive Pluronic F127 gel. Peptides and proteins are pre-stained by remazol brilliant blue (RBB) and loaded by a 10 micro-liter syringe. This method demonstrates semi-quantification ability and good reproducibility for small organic molecules (dyes), peptides (from 210 Da to 6,000 Da and up) and small proteins. The hydrophobic/hydrophilic interaction plays an important role in its separation mechanism. The target Pluronic gel fraction can be easily pushed out of the tube for both qualitative and quantitative analysis by NanoDrop UV-Vis spectrophotometry, high performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization (MALDI/MS) within 20 minutes. The standard curves from NanoDrop UV-Vis and HPLC show around 99% linearity. The recovery of Alizarin Red S from gel fraction is over 90%. HPLC can even lower the detection limit for cytochrome c to 100 ppb with 99% linearity. The conjunction of HPLC with Pluronic tube gel electrophoresis is intended to provide a facile important check for overlapping electrophoresis bands. Pluronic polymers suppress MALDI/MS signals of proteins, not peptides even those pre-stained by RBB. The suppression mechanism includes the hydrophobic/hydrophilic related aggregation and adsorption. Pluronic F127 should be removed from the gel fraction solution by solid phase extraction before MALDI/MS analysis of proteins. However, gel removal is not necessary for MALDI/MS of peptides. The base catalyzed alcohol-glycidyl ether reaction can add quaternary ammonium groups to the Pluronic polymer changing its physical, chemical, and microbiological properties. The separation results of dyes, peptides, and proteins in this modified Pluronic gel have also been altered. A five peptide mixture pre-stained by RBB cannot be separated in the unmodified Pluronic gel, but can be separated into three bands with the modified Pluronic polymer with identification by MALDI/MS. The modification confirms the hydrophilic (possibly ion exchange) interaction and provides a new Pluronic gel for electrophoresis separation.
Neil D. Danielson, PhD (Advisor)
Andre J. Sommer, PhD (Committee Chair)
Ann E. Hagerman, PhD (Committee Member)
Scott Hartley, PhD (Committee Member)
Paul Harding, PhD (Committee Member)
163 p.
Analytical Chemistry
electrophoresis, wide bore tube, Pluronic gel, uncommon gel, peptides, low molar mass proteins, NanoDrop UV-Vis, HPLC, MALDI MS, dyes, modified Pluronic , polyacrylamide gel electrophoresis, pre-stain, glycidyltrimethylammonium, surfactant suppression

Recommended Citations

Citations

  • Wei, W. (2013). Wide bore tube electrophoresis using Pluronic polymer gels in conjunction with spectrophotometry, HPLC, and MALDI/MS [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1375056442

    APA Style (7th edition)

  • Wei, Wenjun. Wide bore tube electrophoresis using Pluronic polymer gels in conjunction with spectrophotometry, HPLC, and MALDI/MS. 2013. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1375056442.

    MLA Style (8th edition)

  • Wei, Wenjun. "Wide bore tube electrophoresis using Pluronic polymer gels in conjunction with spectrophotometry, HPLC, and MALDI/MS." Doctoral dissertation, Miami University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=miami1375056442

    Chicago Manual of Style (17th edition)

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© 2013, all rights reserved.
This open access ETD is published by Miami University and OhioLINK.