Assays of nucleosome assembly and the inhibition of histone acetyltransferase activity. (6) Reconstitution of chromatin; (7) Analysis of nucleoproteins on an agarose gel; and (8) Purification of reconstituted chromatin on a sucrose gradient
Mononucleosomes can be reconstituted on 197-bp or 147-bp radiolabeled DNA fragments (see Notes 1 and 2) either by salt dialysis using purified core histones or by histone octamer transfer from donor H1-depleted chromatin. Usually, reconstitution yields naked DNA and DNA with mono-, di-, and trinucleosome cores.
The efficiency of reconstitution and the number of nucleosome cores on a labeled DNA fragment can be analyzed by agarose gel electrophoresis.
Reconstituted oligonucleosomes can be separated by sucrose gradient centrifugation on the basis of the number of histone octamers bound to a DNA fragment. We use 5% to 20% (w/v) linear sucrose gradients to isolate dinucleosomes from unreconstituted DNA, free histones, mono- and trinucleosomes.
For a detailed introduction to assays of nucleosome assembly and the inhibition of histone acetyltransferase activity, please go here: "http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly.php":http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly.php
Salt Dialysis Using Purified Core Histones
Then dialyze the mixture against 1 L of TEMP buffer for another 4 h at 4 °C. Keep the reconstituted samples in siliconized tubes on ice until use (see Note 5).
Octamer Transfer from Donor Chromatin (see Note 6)
Dialyze the sample for 4 h against 1 L of TEMP buffer at 4 h and then keep the reconstituted samples in siliconized tubes on ice until use (see Note 5).
Subject the dried gel to autoradiography.
Purification of Reconstituted Chromatin on a Sucrose Gradient
Pool respective fractions that contain mono-, di-, and trinucleosomes separately, concentrate proteins to greater than 5 μg/mL (as protein) using Centricon-30, and dialyze preparations against TEMP buffer at 4 °C to remove sucrose. Store samples in siliconized tubes on ice prior to use (see Note 5).
Binding of Linker Histones to Reconstituted Chromatin
Reconstituted short chromatin is a useful substrate for investigations of the binding of chromatin proteins, for example, linker histones and sequence-specific DNA-binding proteins, to nucleosomal DNA2,3. Native interactions between histone H1 and spaced dinucleosomes can be examined in vitro as follows.
Posted 30 Jul, 2007
Assays of nucleosome assembly and the inhibition of histone acetyltransferase activity. (6) Reconstitution of chromatin; (7) Analysis of nucleoproteins on an agarose gel; and (8) Purification of reconstituted chromatin on a sucrose gradient
Posted 30 Jul, 2007
Mononucleosomes can be reconstituted on 197-bp or 147-bp radiolabeled DNA fragments (see Notes 1 and 2) either by salt dialysis using purified core histones or by histone octamer transfer from donor H1-depleted chromatin. Usually, reconstitution yields naked DNA and DNA with mono-, di-, and trinucleosome cores.
The efficiency of reconstitution and the number of nucleosome cores on a labeled DNA fragment can be analyzed by agarose gel electrophoresis.
Reconstituted oligonucleosomes can be separated by sucrose gradient centrifugation on the basis of the number of histone octamers bound to a DNA fragment. We use 5% to 20% (w/v) linear sucrose gradients to isolate dinucleosomes from unreconstituted DNA, free histones, mono- and trinucleosomes.
For a detailed introduction to assays of nucleosome assembly and the inhibition of histone acetyltransferase activity, please go here: "http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly.php":http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly.php
Salt Dialysis Using Purified Core Histones
Then dialyze the mixture against 1 L of TEMP buffer for another 4 h at 4 °C. Keep the reconstituted samples in siliconized tubes on ice until use (see Note 5).
Octamer Transfer from Donor Chromatin (see Note 6)
Dialyze the sample for 4 h against 1 L of TEMP buffer at 4 h and then keep the reconstituted samples in siliconized tubes on ice until use (see Note 5).
Subject the dried gel to autoradiography.
Purification of Reconstituted Chromatin on a Sucrose Gradient
Pool respective fractions that contain mono-, di-, and trinucleosomes separately, concentrate proteins to greater than 5 μg/mL (as protein) using Centricon-30, and dialyze preparations against TEMP buffer at 4 °C to remove sucrose. Store samples in siliconized tubes on ice prior to use (see Note 5).
Binding of Linker Histones to Reconstituted Chromatin
Reconstituted short chromatin is a useful substrate for investigations of the binding of chromatin proteins, for example, linker histones and sequence-specific DNA-binding proteins, to nucleosomal DNA2,3. Native interactions between histone H1 and spaced dinucleosomes can be examined in vitro as follows.
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