Figure 1.
Histone purification strategies.
Schematic depiction of the workflow of (A) the conventional histone purification method according to Luger and coworkers [2], [3] and (B) our RHP protocol. For further details see the main text. Footnotes indicate variations and simplifications of the initial protocol. * The gel filtration step was successfully omitted in simplified purification schemes [5], [7]–[9]. # These steps can be replaced by dilution into or dialysis against SAU 200 buffer [5]–[9]. ‡ To remove possible DNA contaminations, it was suggested to filter the sample through an anion exchange resin prior to applying it to the cation exchange chromatography [7]–[9]. § Anion exchange filtering and cation exchange chromatography can be combined. See note in step 3.2 and Figure S2 in File S1.
Table 1.
Extinction coefficients of Drosophila histones at 280 nm.
Figure 2.
Whole cell extracts were prepared under denaturing conditions from bacteria expressing Drosophila H2B by French Press and sonication. Cell debris and residual insoluble material were pelleted by centrifugation. Efficiency of the histone extraction was analyzed on Coomassie-stained SDS gels by loading equivalent amounts of the supernatant containing the solubilized histones (SN) and the corresponding pellet fraction (P). Most H2B was present in the supernatant.
Figure 3.
Histone purification by cation exchange chromatography.
The whole cell extract from Figure 2 containing solubilized Drosophila H2B (SN) was filtered and applied to cation exchange chromatography under denaturing conditions. (A) Equivalent amounts of the filtered whole cell extract (Input) and the flow-through fraction of the cation exchange column were analyzed by SDS-PAGE. Most H2B bound to the chromatography resin. (B) H2B was eluted by a NaCl gradient as indicated. Fractions 4–8 were pooled and processed further as described in the main text.
Figure 4.
Side-by-side comparison of histone purities.
Histones were purified according to the RHP protocol (RHP) and according to a published protocol that started with the preparation of inclusion bodies (IB; ref. 5). Both purification procedures started from the same amount of bacteria that were grown on the same day. (A) SDS-PAGE analysis. H2A showed the weakest overexpression (Fig. S1 in File S1) and is consequently the least pure. M: protein marker. (B) Purities.
Figure 5.
Histones purified according to the RHP protocol were assembled into octamers. The elution profile of the size exclusion chromatography column is depicted (upper panel). The protein content of selected elution fractions was analysed by SDS-PAGE (lower panel and Fig. S3 in File S1). Octamers eluted with a tailing shoulder, which contained a contaminating protein (asterisk).
Figure 6.
Quality control of the histone octamers.
Stoichiometry and purity of the octamers assembled from the histones purified according to the RHP method outlined in the main text were analyzed by SDS-PAGE (RHP). An octamer preparation assembled from histones purified from inclusion bodies according to published protocols was loaded in parallel (IB; ref. 5). The asterisk marks a contamination that is present to a lesser extent in RHP octamers.