Application example

10 µg of total protein from Arabidopsis thaliana Col0 (1), Cat2-(Col0) (2), Ler0 (3), Cat2-(Ler0) (4), Zea mays (5), Oryza sativa (6), Brassica oleracea (7), Nicotiana bentamina (8) were extracted with 60mM Tris pH 6.9, 10mM DTT, 20% glycerol, 1mm PMSF were separated on 12.5% SDS-PAGE and blotted 1h to PVDF. Blot was blocked with 3% skim milk in PBS+0.05% Tween20 for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h at RT with agitation in the same buffer. The antibody solution was decanted and the blot was rinsed briefly three times, then washed 3 times for 5 min in PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, Agrisera, AS09 602) diluted to 1:50 000 in 3% skim milk in PBS+0.05% Tween20 for 1h at RT with agitation. The blot was washed as above and developed for 1 min with Western Lightning Plus-ECL ( PerkinElmer )according to the manufacturers instructions. Exposure time was  5min. in ChemiDoc XRS+ (Biorad ).

Courtesy of Brigitte van de Cotte, Gent University, Belgium

Blots were performed from 10 µg of protein from total extracts, crude extracts as well as from isolated tagged-mitochondria (leaves or roots). Arabidopsis thaliana protein extracts were prepared using a protein extraction buffer (100 mM Tris-HCl pH 7.5, 50 mM EDTA, 250 mM NaCl, 0.05% SDS). Samples were denatured with Laemmli buffer (Bio-Rad) supplemented with 10% β-mercaptoethanol at 95°C for 10 min before separating the protein mixtures on reducing 12% polyacrylamide gel. Protein extracts were blotted 1h onto a 0.45 µm nitrocellulose membrane using wet transfer. Blots were blocked with 5% milk for 1h/RT with agitation. Blots were incubated with the primary antibodies anti-catalase (Agrisera, AS09 501) at a dilution of 1: 1 000 for ON/4°C with agitation in TBS-T or PBS-T + 2% milk, respectively. Blots were washed 3 times for 5 min in TBS-T or PBS-T at RT with agitation. Blot was incubated for 1h/RT with agitation in Agrisera matching secondary antibodies (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:10 000 in TBS-T or PBS-T + 2% milk.  Blots were washed 3 times for 5 min in TBS-T or PBS-T following by 3 additional washing steps for 5 min in TBS or PBS. Visualization was carried out using the chemiluminescence kit Agrisera ECLBright; AS16 ECL-N-100 and signals were detected using Azure c600 Western Blot Imaging system (Azure biosystems). Exposure time was 2-5 min.

Courtesy of Dr Jonathan Przybyla-Toscano, Umeå Plant Science Centre, Sweden

To obtain reactivity with Solanum lycopersicum urea gel needs to be apply. Please, contact us for more details.

To decrease background signal this antibody needs to be incubated in PBS-T NOT TBS-T. For reference, check image in application example below.

Catalase is an enzyme found in most living organisms which is catalazying decomposition of hydrogen peroxide to water and oxygen. In plant cells catalase is found in peroxisomes. This enzyme is involved in photorespiration and symbiotic nitrogen fixation.