Abstract
Fluorescein is widely used for protein labeling because of its high extinction coefficient and fluorescence emission quantum yield. However, its emission is readily quenched by various pathways. We exploit these properties of fluorescein to examine the self-association of a DNA binding protein and determine the amount of the protein in gel-shifted complexes with specific DNA. A construct (HSFDT385SH) of the heat shock transcription factor (HSF) was expressed that contains the DNA-binding and trimerization domains, residues 192-385 of HSF, with four additional COOH-terminal residues, GMLC, and then labeled at the COOH-terminal cysteine with fluorescein 5-maleimide to form HSFDT385-Fl. The fluorescence increase accompanying the formation of heterotrimers on titration of HSFDT385-Fl with HSFDT385SH) led to an estimate of 3 x 10(-16) M2 for the equilibrium constant for trimerization of HSFDT385SH. HSFDT385-Fl fluorescence also increased 1.7-fold on binding to specific DNA, but not to nonspecific DNA. The protein and DNA content of the several gel-shifted complexes of HSFDT385-Fl (lambdamaxem 532 nm) with specific DNA labeled noncovalently with the energy transfer heterodimer TOTAB (lambdamaxem 658 nm) were accurately determined by ...Continue Reading
References
Sep 15, 1992·Analytical Chemistry·X C HuangR A Mathies
Jun 11, 1992·Nucleic Acids Research·H S RyeA N Glazer
Jan 25, 1991·Nucleic Acids Research·H S RyeA N Glazer
Jan 1, 1990·Methods in Enzymology·F W StudierJ W Dubendorff
Dec 1, 1989·Cell·P K Sorger, H C Nelson
Oct 1, 1988·Analytical Biochemistry·M Granger-SchnarrM Schnarr
Mar 4, 1988·Science·H Xiao, J T Lis
Sep 1, 1988·Molecular and Cellular Biology·J AminR Voellmy
May 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·W Hendrickson, R Schleif
Dec 11, 1981·Nucleic Acids Research·M Fried, D M Crothers
Nov 23, 1982·Biochemistry·M M Garner, A Revzin
Jan 11, 1983·Nucleic Acids Research·M G Fried, D M Crothers
Nov 25, 1983·Nucleic Acids Research·A KolbH Buc
Jan 1, 1993·Analytical Biochemistry·H S RyeA N Glazer
Dec 11, 1993·Nucleic Acids Research·S C BensonA N Glazer
Dec 11, 1993·Nucleic Acids Research·S C BensonA N Glazer
Jan 1, 1993·Methods in Enzymology·H S RyeA N Glazer
Citations
Oct 13, 2001·Proteins·O Littlefield, H C Nelson
Apr 6, 2011·Biochemistry·Cesar KenaanPaul F Hollenberg
Oct 2, 2002·Acta Physiologica Scandinavica·M J HorterA Von Eckardstein
Apr 9, 2001·Nucleic Acids Research·M P CiceroH C Nelson
Dec 17, 2009·Nucleic Acids Research·Richard D HuttonJ Carlos Penedo
Dec 7, 2007·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·Jonathan G BoucherDaniel L Sparks
Feb 1, 2009·Clinical Lipidology·Mary G Sorci-ThomasMichael J Thomas
Aug 10, 2000·Analytical Biochemistry·J Wildeson, C J Murphy
Jan 18, 2011·Analytica Chimica Acta·Meng Jing, Michael T Bowser
Dec 20, 2007·BMC Medical Genetics·Zari DastaniMichel Marcil
Aug 14, 1998·Molecular Cell·M ZhongC Wu
Jun 26, 2004·Current Opinion in Cardiology·Michael Miller, Min Zhan
Nov 5, 2013·Current Opinion in Lipidology·Brian R Krause, Alan T Remaley
Feb 28, 2003·Human Genetics·Ching Yin LeeMichel Marcil
Mar 25, 2009·Current Opinion in Endocrinology, Diabetes, and Obesity·Xavier RoussetAlan T Remaley
Jul 28, 2011·Electrophoresis·Soo Hyun ParkYoung Sook Yoo
Apr 11, 2009·Langmuir : the ACS Journal of Surfaces and Colloids·Jasper O HardestyCurtis W Frank
Nov 21, 1997·Journal of Molecular Biology·B L DreesH C Nelson
Nov 5, 1997·Analytical Biochemistry·Z ZengA N Glazer
May 2, 1998·Biochimica Et Biophysica Acta·D L SparksT A Neville
Mar 26, 1999·Biochemistry·R PeteranderlH C Nelson
Jul 29, 2000·Biochemistry·E R Jamieson, S J Lippard