SAM1-4 | S-adenosylmethionine synthase

43.2 | 45 kDa (Arabidopsis thaliana)

Courtesy of Louis-Valentin Meteignier, LGBP, Faculté des Sciences de Luminy, France

Leaf tissue of Arabidopsis thaliana Col-8 plants was disrupted via mortar and pestle under liquid nitrogen. Leaf protein was extracted by addition of appropriate amounts of extraction buffer (6 M Guanidine-HCl, 100 mM HEPES, 5 mM EDTA and 1x Halt™ Protease Inhibitor-Cocktail). Extracted protein was chloroform/methanol-precipitated and resolved in 2 % SDS, 50 mM Tris (pH 7.4). Protein concentration was determined using BCA assay. 100 µg of cleaned protein were mixed with 4x LDS sample buffer (final conc. 1x), 50 mM DTT (final) and heated 2x to 95 °C for 5 minutes for denaturation. 2.5 µg of protein were loaded per lane on a 12 % SDS PAGE gel. After separation, proteins were blotted to a PVDF membrane for 30 minutes with 25 V and 1 A in a semi-dry blot. Blots were blocked with 5 % Milk in TBS (= Tris buffered saline) for 2 h at room temperature. Blots were subsequently washed with TBS-T (TBS + 0.05 % Tween20) for 1h at room temperature with 3 exchanges of wash buffer. Primary antibody (anti SAM1-4) was used at a dilution of 1:2000 in 1 % Milk in TBS-T and blots were incubated over night at 4 °C with gentle agitation. Primary antibody was decanted and blots were again washed for 1 h with TBS-T as mentioned above. Secondary antibody (anti Rabbit IgG, HRP conjugated) was used at a dilution of 1:20000 in 1 % Milk in TBS-T and blots were incubated for 1 h at room temperature. After decanting of secondary antibody, blots were again washed with TBS-T for 1h. Before Chemiluminescence detection, blots were shortly washed with TBS without Tween20 3x 5 minutes at room temperature. Chemiluminescent detection reagent was used for signal detection. Images of the blots were obtained using ChemiDoc™ XRS (Bio-rad) in high resolution mode. Exposure time was 20 seconds.

Courtesy of phd student Andreas Perrar, Prof. Dr. Pitter Huesgen group, Forschungszentrum Jülich, Germany