In the context of sodium carbonate transfer we found that PVDF membranes yielded more reproducible effects for detecting actually subtle differences in protein levels than did the use of nitrocellulose membranes (Figure 3, compare lanes 1 C 4 with 5 C 8). aliquots at ?20 F2RL1 C. Just before use add -mercaptoethanol to 5 % (v/v) final concentration. For sodium carbonate transfer: 40 sodium carbonate transfer buffer, pH 9.5 For 1 l:21.1 g NaHCO3 (251 mM), 18.35 g Na2CO3 (173 mM).Blend in 0.9 l increase distilled water (ddH20), pH to 9.5 with 10 N NaOH, add ddH20 to 1 1 l. Store at RT. Open in a separate windows For Tris-glycine transfer: 20 Tris-glycine transfer buffer For 1 l: 24.2 g Tris MK-5108 (VX-689) foundation (200 mM), 150.1 g glycine (2 M). Blend in 1 l double distilled water (ddH20). pH adjustment not necessary (will become ~8.8). Store at RT. 100 % methanol Ponceau-S answer (0.5 % [w/v] Ponceau S, 1 % [v/v] glacial acetic acid) Destaining solution (0.1 % [v/v] glacial acetic acid) 10 TBS For 1 l:30 g Tris base (248 mM), 80 g NaCl (1.37 M), 2 g KCl (27 mM).Blend in 0.9 l ddH20, pH to 7.4 with concentrated HCl, add ddH20 to 1 1 l.Store at RT. Open in a separate windows 1 TBST (1 TBS, 0.1 % Tween 20) Dry milk (e.g., CARNATION NonFat Dry Milk, Nestl, Glendale, CA) Main antibody, e.g., anti-H3K4me3 (abdominal8580, Abcam, Cambridge, MA) Secondary antibody, e.g., anti-rabbit IgG, horseradish peroxidase(HRP)-linked (NA934V, GE Healthcare, Piscataway, NJ) Chemiluminescent substrate for detection of HRP conjugates (SuperSignal Western Pico chemiluminescent substrate, Thermo Scientific, Rockford, IL) Products Chromatography paper (Grade 3MM Chr, Whatman) Gel electrophoresis apparatus, space heat Polyvinyldifluoride (PVDF) transfer membrane (Immobilon P, pore size 0.45 m, Millipore) Millipore also offers Immobilon-PSQ membranes for proteins in the range of 10 C 20 kDa, in MK-5108 (VX-689) case Immobilon-P membranes do not yield optimal results Amersham Hybond-P (GE Healthcare, pore size 0.45 m) also yields good results Electroblotting apparatus, space temperature Clean box(s) for immunoblot Plastic wrap (e.g., Saran Wrap) Shakers, orbital or platform, space heat Darkroom and X-ray film programmer Method Whole cell protein components from candida and electrophoresis 1 Collect on the subject of 10 OD600 equivalents of cells (e.g., 20 ml of a tradition at OD600 of 0.5) from a logarithmically growing MK-5108 (VX-689) liquid tradition into 15-ml conical-bottom tube by spinning at 3,750 g for 5 min at 4 C. 2 Pour off supernatant and wash once with ice-cold ddH20. 3 Pour off supernatant, transfer pellet with rest of supernatant into 1.5-ml microcentrifuge tube and pellet by spinning at 20,800 g for 10 s at 4 C. 4 Pipette off supernatant. This is to avoid loosing cells leading to uneven loading of gel. Can freeze pellet in liquid nitrogen or on dry ice. Store at ?70 C. Thaw pellet on snow. 5 Resuspend cell pellet in 100 l of ddH20, then add 300 l of 0.2 M sodium hydroxide solution and 20 l -mercaptoethanol. 6 Incubate the sample for 10 min on snow. 7 Pellet the sample inside a microcentrifuge at 20,800 g for 10 min at 4 C. 8 Resuspend the pellet in 100 l of 1 1 SDS-PAGE sample buffer. 9 Boil the sample for 10 min and pellet inside a microcentrifuge at 10,600 g for 3 min at space temperature. 10 Use 6 C 12 l of the supernatant per lane of a 15 % SDS-polyacrylamide gel and handle by electrophoresis (observe SDS-PAGE of proteins). Protein transfer to membrane 11 After electrophoresis, transfer proteins in a tank of buffer relating to Immunoblotting: Submerged Electrophoretic Transfer of Proteins from Gels to Membranes with the following exceptions/notes: 12 Use PVDF membrane prepared as per manufacturers instructions – for Immobilon-P: Soak in 100 % methanol for 15 s; Wash in ddH20 for 2 min; Equilibrate in transfer buffer for 5 min. 13 Protein transfer conditions depend on transfer buffer used: 1 sodium carbonate transfer buffer, 20 % methanol for protein transfer. Transfer at 0.5 A (fixed) for 60 min at room temperature. Alternative transfer conditions might be possible (e.g., at 22 V [fixed], for 90 min at space temperature, see conversation). 1 Tris-glycine transfer buffer,.