Protein Protocols Protein Protocols

Western Blotting of Basic Proteins 339
Fig. 1. Electrophoretic transfer of histones from AUT minislab gels. (A) Histones (9, 18, and
36 µg in lanes a, b, and c, respectively) isolated from T-47D-5 human breast cancer cells were
electrophoretically resolved on AUT minislab gels. The gel was stained with Coomassie blue.
(B) The Coomassie blue-stained AUT minislab gel pattern of histones remaining after transfer
(30 V overnight) to nitrocellulose is shown. (C) The India ink-stained nitrocellulose pattern of
histones transferred from the AUT minislab gel in B is shown. A 0, A 1, and A 2 correspond to the
un-, mono-, and diacetylated species of histone H4, respectively. The ubiquitin adduct of histone
H2A is denoted as uH2A. Reprinted with permission from ref. 10 (copyright by the Academic
Press).
2. The India ink stain is added to the nitrocellulose membrane (0.56 mL/cm 2 ) which is agitated
for 30 min to 2 h at room temperature.
3. The stain is discarded, and the nitrocellulose membrane is shaken in water for 5 min at
room temperature.
4. The nitrocellulose membranes are dried and stored.
4. Notes
1. Nitrocellulose membranes have been used in the majority of our studies. However, these
membranes are fragile and must be handled with care. An alternate membrane, which is
stronger than nitrocellulose, is PVDF (Bio-Rad). The PVDF membranes are wetted with
100% methanol for 3 min and then equilibrated with transfer buffer for 3 min.
2. The efficiency of transfer of basic proteins (histones) from AUT polyacrylamide slab gels
to nitrocellulose membranes is shown in Fig. 1. Most of the histones were efficiently
transferred. The efficiency of elution was poorest for histone H1. Figure 1C demonstrates
that the histone variants of histone H2A (H2A, H2A.Z) and of histone H3 (H3.1, H3.2,
H3.3) and the modified histone species (e.g., ubiquitinated histone H2A, acetylated histone
H4) were transferred. Densitometric tracings of the gel patterns before and after transfer
demonstrated that >90% of the histones H2A, H2B, H3, and H4, and approx 80% of
histone H1 were eluted from the AUT gel.