Separation of Histone Variants and Post‐Translationally Modified Isoforms by Triton/Acetic Acid/Urea Polyacrylamide Gel Electrophoresis

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Due to their similarities in size and charge, complete resolution of histones by electrophoresis poses a considerable challenge. The addition of nonionic detergents to the traditional acetic acid/urea (AU) polyacrylamide gel electrophoresis (PAGE) system has afforded an excellent method to separate not only the different modified forms of histones, but also the primary sequence variant subtypes of selected histone species; it is widely used to separate histones with varying levels of acetylation. This unit describes the use of gels containing the nonionic detergent Triton X?100, referred to as Triton/acetic acid/urea (TAU) polyacrylamide gels, for analysis of histones. Also included are support protocols detailing several accessory techniques: assembly of gel plates for the TAU gel, preparation of histones from isolated nuclei in a solubilized form amenable to electrophoresis, and electrophoretic transfer of proteins from these gels to PVDF membranes.

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Basic Protocol 1: Triton/Acetic Acid/Urea (TAU) Polyacrylamide Gel Electrophoresis for Analysis of Histones
Support Protocol 1: Assembly of Gel Plates
Support Protocol 2: Histone Isolation from Prepared Nuclei
Support Protocol 3: Electrophoretic Transfer of TAU‐Polyacrylamide Gels
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Triton/Acetic Acid/Urea (TAU) Polyacrylamide Gel Electrophoresis for Analysis of Histones

Materials

Glacial acetic acid (17.4 M)
TEMED (N,N,N′,N′‐tetramethylethylenediamine )
recipe9.5 M urea (see recipe )
10% (w/v) ammonium persulfate (APS; prepare fresh for each gel)
10% Triton X‐100 (protein grade; Calbiochem)
recipe63.5% acrylamide/0.4% bisacrylamide (see recipe )
Moderately warm water (∼45°C) in a beaker
recipeRunning buffer (0.9 M acetic acid ; see recipe )
recipeScavenger solution (2‐mercaptoethylamine ; see recipe )

1000‐ and 100‐ml sidearm flasks
Gel‐casting assembly (see protocol 2 )
Vertical gel electrophoresis apparatus to accommodate 16 × 22–cm glass plates (see protocol 2 )
Power supply capable of running in constant current mode, with two leads
1.5‐mm‐thick 16‐well gel comb
Disposable syringes with 23‐G needles
Disposable syringe with 18‐G needle bent into a U shape
Hamilton syringe

Additional reagents and equipment for degassing gel solutions (unit 10.2 )

Support Protocol 1: Assembly of Gel Plates

Materials

95% ethanol
1% (w/v) agarose, melted
16 × 22–cm notched glass plate

16 × 22–cm glass plate
Two 22‐cm‐long × 1.5‐mm‐thick Teflon spacers
18‐cm‐long × 1.5‐mm‐thick Teflon spacer
Six 5‐cm‐wide stainless steel binder clips (Research Products International)
Resealable plastic bag large enough to contain gel‐casting assembly

Support Protocol 2: Histone Isolation from Prepared Nuclei

Materials

Isolated nuclei (e.g., see unit 12.1 )
Concentrated sulfuric acid (H₂ SO₄ ; 18 M H₂ SO₄ ) diluted to 0.2 M
100% trichloroacetic acid (TCA)
11.6 M hydrochloric acid (HCl)
100% acetone
recipeAcetic acid/urea sample buffer (see recipe )

Support Protocol 3: Electrophoretic Transfer of TAU‐Polyacrylamide Gels

Materials

recipeWash buffers I and recipeII (see reciperecipes )
recipeCAPS transfer buffer (see recipe )
100% methanol
Ponceau S stain (Sigma; optional)
Transfer membrane (e.g., Immobilon‐P PVDF membrane, Millipore)
Filter paper cut to size of gel

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Figures

Figure 21.2.1 HeLa cells were synchronized with a double thymidine block procedure (2.7 mM thymidine; Peterson and Anderson, ; Knehr et al., ) and released into S phase for 3 hr. Released cells were then incubated 60 min in the absence (−) or presence (+) of okadaic acid (500 nM; Calbiochem), an inhibitor of cellular phosphatases. Acid‐soluble nuclear proteins and isolated histone H1 were subjected to electrophoresis in a TAU gel and analyzed by Coomassie blue staining. Lane M contains hyperphosphorylated H1 (pH1) from cells arrested in M phase by treatment with Colcemid (demecolcine; 0.06 µg/ml). The un‐ (Ac 0) and monoacetylated (Ac 1) isoforms of histone H4 are indicated.

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Figure 21.2.2 HeLa cells were synchronized with a double thymidine block procedure and released into S phase for 4 hr (lane S). Isolated histone H1 was subjected to electrophoresis in a TAU gel; lane M contains hyperphosphorylated H1 from cells arrested in M phase by treatment with Colcemid (demecolcine). The gel was transferred electrophoretically to Immobilon‐P membrane (Millipore) and probed with antibodies specific for either phosphorylated H1 (a gift of Dr. C. David Allis) or total H1. Bound antibodies were detected by a secondary alkaline phosphatase color reaction. Bullets indicate the positions of the unphosphorylated forms of H1, as determined by staining the membrane with Ponceau S. The mono‐ (1), di‐ (2), tri‐ (3), and hyperphosphorylated (H) isoforms of H1 are indicated.

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Literature Cited

	Delcuve, G.P. and Davie, J.R. 1992. Western blotting and immunochemical detection of histones electrophoretically resolved on acid‐urea‐triton and sodium dodecyl sulfate‐polyacrylamide gels. Anal. Biochem. 200:339‐341
	Franklin, S.G. and Zweidler, A. 1977. Non‐allelic variants of histones 2a, 2b, and 3 in mammals. Nature 266:273‐275
	Knehr, M., Poppe, M., Enulescu, M., Eickelbaum, W., Stoehr, M., Schroeter, D. and Paweletz, N. 1995. A critical appraisal of synchronization methods applied to achieve maximal enrichment of HeLa cells in specific cell cycle phases. Exp. Cell Res. 217:546‐553
	Mizzen, C.A. and Allis, C.D. 1998. Linking histone acetylation to transcriptional regulation. Cell. Mol. Life Sci. 54:6‐20.
	Panyim, S. and Chalkley, R. 1969. High resolution acrylamide gel electrophoresis of histones. Arch. Biochem. Biophys. 130:337‐346
	Peterson, D.F. and Anderson, E.C. 1964. Quantity production of synchronized mammalian cells in suspension culture. Nature 203:642‐643
	van Holde, K.E. 1988. Chromatin. Springer‐Verlag, New York.
	Waterborg, J.H. and Harrington, R.E. 1987. Western blotting of histones from acid‐urea‐triton‐ and sodium dodecyl sulfate‐polyacrylamide gels. Anal. Biochem. 162:430‐434
	Wolffe, A.P. 1995. Chromatin. Academic Press, San Diego.
	Zweidler, A. 1978. Resolution of histones by polyacrylamide gel electrophoresis in presence of nonionic detergents. Methods Cell Biol. 17:223‐233
Key References
	Zweidler, 1978. See above.
	Initial description of Triton/acetic acid/urea polyacrylamide gel electrophoresis.
	Delcuve and Davie, 1978. See above.
	Description of the development of a method for efficiently transferring the Triton/acetic acid/urea polyacrylamide gels to membrane.