Anti ha hrp clone 3f10

Proteins were separated by electrophoresis on 8% or 10% 29:1 polyacrylamide gels in 1× TGS buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) and transferred onto nitrocellulose membranes using the Bio-Rad TransBlot system. Membranes were saturated by 30 min incubation in TBS-T buffer (Tris-buffered saline with 0.1% Tween 20), containing 5% (w/v) powder milk. Specific proteins were detected by incubation in TBS-T with the following antibodies: anti-HA-HRP (Clone 3F10, Roche) diluted 1000-fold; anti-Dbp6 polyclonal antibodies diluted 10 000-fold (36 (link)); anti-PGK1 (clone 22C5D8, Invitrogen) diluted 10 000-fold; rabbit peroxidase anti-peroxidase soluble complex (PAP, Sigma) diluted 10 000-fold. After incubation with primary antibodies, membranes were washed three times with TBS-T. Anti-rabbit or mouse IgG–HRP conjugate (Promega) were used, when needed, as secondary antibodies. After three washes of the membranes with TBS-T, proteins associated with antibodies were detected as chemiluminescent signals using Clarity Western ECL Substrate (Bio-Rad) and the ChemiDoc imager apparatus (Bio-Rad) followed by quantification with the ImageLab software (Bio-Rad).

The wheat germ-based ubiquitination assays of RING proteins were carried out as previously described [25 (link)]. The assays were performed in a 10 μL reaction mixture containing 20 mM Tris-HCl pH 7.5, 0.2 mM DTT, 5 mM MgCl₂, 10 μM zinc acetate, 3 mM ATP, 1 mg/mL BSA, 400 nM human recombinant FLAG-Ub or HA-Ub (Boston Biochem, http://www.bostonbiochem.com), 1 μL recombinant E2, and 1 μL recombinant RING protein at 37 °C for 3 h. The reactions were terminated by the addition of 5 μL 3× SDS sample buffer and boiling for 5 min, and were then analysed on 5–20 % SDS-PAGE followed by immunoblotting using anti-FLAG-HRP (Sigma) or anti-HA-HRP (clone 3 F10) antibodies (Roche Life Science).

Protein samples were incubated with 2x sample buffer [Tris-HCl 250 mM, pH 6.8; glycerol 20% (v/v); SDS 4% (p/v); β-mercaptoethanol 10% (v/v) and bromophenol blue 0.025% (p/v)] for 5 min at 65°C, immediately after extraction. 30 μl of the protein samples were run in an 8% SDS-PAGE gel and blotted on a 0.45 μm nitrocellulose membrane (Amersham Protran) at 4°C. The membrane was stained with 0.1% Ponceau S in 1% acetic acid for 5 min. The background stain was removed by treatment with 1% acetic acid during 15 min before imaging. The Ponceau S was completely removed with T-TBS buffer [Tris-HCl 20 mM, pH 7.5; NaCl 0.5 M; Tween-20 0.1%]. The membrane was probed as previously indicated [42 (link)], with 1:100 anti-HA-HRP (clone 3F10 Roche) or 1:100 anti-GFP-HRP (A10260 Invitrogen) in T-TBS, and developed by a colorimetric assay (0.5 mg/ml DAB in 0.1 M imidazole pH 7 with 0.1 μl/ml 30% H₂O₂ and 54 μl/ml 0.6% CoCl₂) [47 (link)]. Both antibodies were checked consecutively on the same membrane with a washing step (T-TBS buffer for 30 min) in between. The PageRule pre-stained protein ladder was used as size marker of 170, 130, 100, 70 (red), 55, 40, 35, 25 and 15 kDa (Thermo Scientific CN26616). The respective molecular weights are: SLO2-HA, 84.03 kDa; DYW2-GFP, 92.56 kDa; MEF57-GFP, 100.62 kDa; and HSP60.3B-GFP, 87.42 kDa.Image J software was used to quantify the band intensity.