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Development of analytical techniques...

Yang, Peilin.

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Development of analytical techniques for protein binding kinetics and drug screening.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Development of analytical techniques for protein binding kinetics and drug screening./
Author:	Yang, Peilin.
Description:	149 p.
Notes:	Adviser: Robert T. Kennedy.
Contained By:	Dissertation Abstracts International68-08B.
Subject:	Chemistry, Analytical. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3276334
ISBN:	9780549181125

Development of analytical techniques for protein binding kinetics and drug screening.
Yang, Peilin.

Development of analytical techniques for protein binding kinetics and drug screening. - 149 p.

Adviser: Robert T. Kennedy.

Thesis (Ph.D.)--University of Michigan, 2007.

These studies have demonstrated the capability of CE for quantitative measurement of affinity interactions, kinetics and screening.

ISBN: 9780549181125Subjects--Topical Terms:

586156
Chemistry, Analytical.

Development of analytical techniques for protein binding kinetics and drug screening.
LDR:03422nam 2200313 a 45 001 939308
005 20110512
008 110512s2007 ||||||||||||||||| ||eng d
020 $a 9780549181125
035 $a (UMI)AAI3276334
035 $a AAI3276334
040 $a UMI $c UMI
100 1 $a Yang, Peilin. $3 1263304
245 1 0 $a Development of analytical techniques for protein binding kinetics and drug screening.
300 $a 149 p.
500 $a Adviser: Robert T. Kennedy.
500 $a Source: Dissertation Abstracts International, Volume: 68-08, Section: B, page: 5197.
502 $a Thesis (Ph.D.)--University of Michigan, 2007.
520 $a These studies have demonstrated the capability of CE for quantitative measurement of affinity interactions, kinetics and screening.
520 $a Cellular chemistry is controlled by affinity interactions between biomolecules. Quantitative analysis of such interactions is important for understanding cellular chemistry and developing drugs and chemical probes. Capillary electrophoresis (CE), as a valuable approach to binding studies, allows multiple sample components including bound and free species to be separated under electric field. In this work, we have developed CE-based methods for analysis of Src homology 2 (SH2) domain protein binding systems.
520 $a A Fluorescence anisotropy CE (FACE) method was developed for the detection of SH2-Bbeta under equilibrium conditions. A fluorescein labeled peptide containing the SH2 domain recognition sequence was used as the affinity probe and was incorporated in the electrophoresis buffer. Besides purifed SH2 domain, the FACE assay was also applied to cell lysate containing full length SH2-Bbeta. For kinetic measurements, two CE-based methods were developed, compared and evaluated using Fyn and SH2-Bbeta as models. Complex half-life (t1/2 ) was measured in either competitive mode with unlabeled ligand spiked in sample and competition monitored by CE, or noncompetitive mode with dissociation intentionally allowed by adjusting the separation time and t1/2 extracted from peak shapes. By optimizing the injection and separation conditions, t1/2 ranging from 0.7 to 50 s were measured.
520 $a The competitive CE assay was also explored for possible drug screening applications using SH2 domain proteins as models as they are interesting pharmaceutical targets. In a multiplexed high-speed CE assay, three SH2 domain proteins, Fyn, Src and SH2-Bbeta, were separated and inhibitors screened against these proteins simultaneously. A non-specific and a specific inhibitor were identified and IC50s were quantitatively determined from peak height of the complex detected at different inhibitor concentrations. Another screening technique developed is on-line HPLC-APCE, which has potential application in screening of effective components from complex mixtures. Three potential peptide inhibitors were separated by HPLC and change in complex peak heights was observed when peptides that interfered with binding eluted from the column. A chromatogram was reconstructed using the peak heights from series electropherograms collected by CE every few seconds.
590 $a School code: 0127.
650 4 $a Chemistry, Analytical. $3 586156
650 4 $a Chemistry, Biochemistry. $3 1017722
690 $a 0486
690 $a 0487
710 2 $a University of Michigan. $3 777416
773 0 $t Dissertation Abstracts International $g 68-08B.
790 $a 0127
790 1 0 $a Kennedy, Robert T., $e advisor
791 $a Ph.D.
792 $a 2007
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3276334