Salt Dialysis Using Purified Core Histones
1. Mix 5 μg of DNA (50-500 ng of radiolabeled DNA and unlabeled carrier DNA) and 5 μg of core histones in 75 to 100 μL of TEMP buffer prepared with 2M NaCl on ice, at a histone-to-DNA ratio (w/w) of 1:1 (see Notes 3 and 4).
2. Transfer the mixture to a dialysis bag and dialyze at 4 °C against 1 L of TEMP buffer that contains NaCl as follows: 2 M NaCl, overnight; 1.5 M NaCl, 1 h; 1 M NaCl, 4 h; 0.75 M NaCl, 4 h; and without NaCl, overnight.
3. 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)
1. Mix 50-500 ng of radiolabeled DNA fragments with H1-depleted donor chromatin at a chromatin DNA-to-fragment DNA ratio (w/w) of from 50:1 to 100:1 in 80 μL of TEMP buffer. Slowly adjust the concentration of NaCl to 1 M with 20 μL of 5 M NaCl.
2. Incubate the mixture at 37 °C for 20 min and then transfer it to a dialysis bag. Dialyze it at 4 °C against 1 L of TEMP buffer that contains 1 M NaCl for 4 h and then against 1 L of TEMP buffer that contains 0.75 M NaCl for 4 h.
3. Then dialyze the sample against TEMP buffer overnight at 4 °C.
4. 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).

Analysis of Nucleoproteins on an Agarose Gel
1. Load samples in 3% (v/v) glycerol without dye onto a 0.7% agarose gel that is submerged in 2 L of 0.5x TBE.
2. Perform electrophoresis at room temperature, at 130 V, for 3 to 4 h (at less than 60 mA; see Note 7). After electrophoresis, put the gel on two sheets of 3MM paper (Whatman Co., Middlesex, UK) and start drying the gel without heating. Turn on the heater of the gel dryer after flattening the gel.
3. Subject the dried gel to autoradiography.

Purification of Reconstituted Chromatin on a Sucrose Gradient
1. Prepare 12-mL 5% to 20% (w/v) linear sucrose gradients using a gradient former or Gradient Master (see Note 8) at room temperature. Cool gradients at 4 °C and store for up to a few hours prior to use.
2. Load samples (less than 300 μL) on gradients and centrifuge at 36,000 rpm for 18 h at 4 °C in an SW41Ti rotor (Beckman).
3. Fractionate the gradients by pumping out from the bottom. The fraction collector should be programmed to collect 300-μL fractions. Analyze each fraction (10 μL of each) by electrophoresis on a 0.7% agarose gel.
4. 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 DNA^2,3. Native interactions between histone H1 and spaced dinucleosomes can be examined in vitro as follows.
1. Incubate dinucleosomes (20 ng of DNA) with various amounts of purified histone H1 in 10 μL of binding buffer at 25 °C for 20 min⁴.
2. Analyze the binding of histone H1 by loading samples directly onto a 0.7% agarose gel and subsequent electrophoresis. The gel can be dried and subjected to autoradiography.