Loading buffer: Roti®-Load, stock solution 4X concentrated (Roth).
Chemiluminescence reagents: Amersham ECL™ Western Blotting Detection Reagents (GE Healthcare); Amersham ECL Plus™ Western Blotting Detection Reagents (GE Healthcare).
Coomassie solution: 0.4g Coomassie brilliant blue G250 dissolved in 200ml of 40% (v/v) methanol in water.
Destaining solution (v/v ratio): 10% acidic acid, 30% methanol, 60% milliQ water. E1 solution: 90mM KCl, 50mM Tris, 5mM MgCl, 0.1mM EDTA, 10mM Na-Butyrat, 0.4mM PMSF, 0.025mM Leupeptin, 2mM DTT; pH 7.4; the solution is filtered, aliquoted in 50ml aliquots and stored at -20°C.
E1 solution/0.25M Sucrose: E1 solution supplemented with 0.25M Sucrose.
E1 solution/0.25M Sucrose/0.5% Triton-X/ 0.5% NP-40: E1 solution supplemented with 0.25M Sucrose, 0.5% Triton-X and NP-40.
E1 solution/1.25M Sucrose: E1 solution supplemented with 1.25M Sucrose.
IP buffer: 100mM NaCl; 10mM Tris; 0.5% NP-40. The pH was adjusted at 7.5; the solution was stored at 4 °C. Complete IP buffer contains also 1mM NaF, 20mM beta- glycerol, 0.1mM NaV, 1mM PMSF and 1 tablete/25ml solution of complete EDTA free protease inhibitor cocktail tablets (Roche).
3.8.2 Preparation of Xenopus laevis whole embryo lysates for SDS-PAGE 25 embryos per condition were collected in 15ml Eppendorf tubes. The embryos were lysed in 100µl of complete IP-Buffer (0.25 embryo equivalent/µl). Samples were centrifuged 15min at 14000rpm at 4°C. 90µl of the supernatant (22.5 embryo equivalent) were transferred to a new 1.5ml eppendorf tube and 18µl of loading dye were added to each sample (the solution contained now 0.21 embryo equivalent/µl). Samples were boiled at 95°C for 5min before loading the gels with 20µl (ca. 4.2
embryo equivalent) from each sample. The rest of samples were snap-frozen in liquid nitrogen and stored at -80°C for repeated use.
3.8.3 Histone extraction from nuclei of Xenopus embryos on sucrose cushion for SDS-PAGE
Sixty embryos at NF30 (Morpholino injection) or at NF11.5 (protein overexpression) were harvested and washed with E1 solution/0.25M Sucrose via centrifugation at 600rpm for 1min. Embryos were incubated for 15-20min at room temperature in the same solution and subsequently lysed after homogenization with 20 strokes using a 5ml glass-glass douncer (Braun, Melsungen). Nuclei were prepared by centrifugation at 1000rpm for 10min at 4°C. The supernatant containing the cytoplasmatic fraction was discarded and the nuclear pellets were resuspended in 3ml of E1 solution/0.25M Sucrose/0.5% Triton-X/ 0.5% NP-40. Nuclei were incubated on ice for 20min. The solution was carefully layered on top of a 50ml falcon containing 5ml of E1 solution/1.25M Sucrose, in order to create two separate phases. Samples were centrifuged for 30min at 2000rpm (NF30 embryos) or 1000rpm (NF11.5 embryos) at 4°C; the solution was discarded and the nuclei were resuspended in 1ml of E1 solution and transferred in 1.5ml Eppendorf tube. Nuclei were centrifuged at 5000rpm for 2min and the pellets were resuspended in the appropriate SDS-loading dye volume (2.5µl loading dye/embryo). Unless used, the samples were kept at -20°C. Before loading the gels, the samples were boiled at 95°C for 5min. Samples obtained from NF30 embryos were diluted 1:10 with loading dye in a new 1.5ml eppendorf tube. The samples were boiled again at 95°C for 5min and finally 15µl of each sample (corresponding approx. to 0.6 (NF30) or 6 (NF11.5) embryo equivalents, respectively) were loaded onto the gels.
3.8.4 Myc-tagged fusion protein extraction from embryos
25 embryos per condition were lysed in 100µl of 100mM NaCl, 10mM Tris pH 7.5 buffer supplemented with 1mM NaF, 20mM beta-glycerol, 0.1mM Sodium Vanadate, 10mM Na Butyrate, 0,5% NP-40 and EDTA-free protease inhibitor cocktail tablets (Roche). Embryos were centrifuged 15min at 14,000g at 4°C; the supernatant was collected and 2X Loading buffer (Roti-Load1; Roth) was added. Samples were subsequently analysed by western blot.
3.8.5 SDS-PAGE and Western Blot Analysis
SDS-PAGE (SDS-polyacrylamide gel electrophoresis) and Western Blot analysis were carried out according to standard protocols (Sambrook et al., 1989), with 10% and 15% PAA gels, using Roti PVDF membrane (Roth). The signals were detected with Amersham ECL™, ECL Plus™ Western Blotting Detection Reagents (GE Healthcare) and with the Infrared Imaging System Li-Cor (Odyssey). When ECL detection reagents were used, the membranes were exposed to several Super-RX Fuji medical X-ray films at different exposure times. When the Infrared Imaging System was used, wet membranes were scanned; several scans were recorded, with different sensitivities. Odyssey Application Software Version 3.0 (Odyssey) was used to quantify the western blot bands intensities.
3.8.6 Sample preparation for Mass Spectrometry
After SDS-PAGE separation, the gels were incubated o/n at 4 °C with Coomassie solution and destained the following day for 4h with frequent exchange of destaining solution every 30-40min. H3 and H4 bands were excised and cut in small pieces. These samples were washed twice with 200µl of H2O on a shaker at 37°C for 5min; neutralization was achieved by incubating the gel pieces with Ammoniumbicarbonate (Ambic) and afterwards a destaining step was performed by incubating the samples with 0.1M Ambic and HPLC-grade Acetonitrile (ACN) on a shaker at 37°C for 30- 90min, followed by additional washes with H2O. Gel pieces were dehydrated by a further ACN incubation. To convert free amino groups to propionic amides of lysine residues, histones were chemically modified by treatment with propionic anhydride before trypsin digestion (0.2µg/µl in 0.1M Ambic). To purify the samples from salts and acrylamide contaminations, the peptide solution was passed over a tip containing small amounts of C18 reversed phased material (ZipTip, Millipore). The peptides were subsequently eluted in a buffer containing 0.1% trifluoracetic acid (TFA), 50% ACN (elution buffer) and spotted on a target plate. The target plate was loaded into a Voyager DE STR spectrometer and spectra were analysed using the Data Explorer software and an in-house-developed software Manuelito. Spectra were de-isotoped and calibrated internally using the autoproteolytic peptides of trypsin. For quantification of the different post-translational modifications (PTMs) of the various peptides obtained after digestion, the relative intensities of each PTM were taken into account. The area under the peak, representing relative intensities of the
modification, was used for quantification. The area of all the modifications for each peptide were summarized and the percentage of each modification was then calculated. Sample preparation and PTM quantifications were performed in collaboration with Tobias Schneider (Laboratory of Professor R. Rupp, Department of Molecular Biology, Adolf Butenandt Institute, LMU, Munich).