PPH1/TAP38 | Protein phosphatase 1
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0,5-5 µg of chlorophyll per lane from Arabidopsis thaliana leaves WT (1-4) and delta-TAP38 (5-8), ecotype Columbia were extracted according to Järvi et al 2016 (Plant Physiol 171:1333-1343) and denatured with SDS and 5% B-mercaptoethanol at 65°C for 10 min. After spinning down the proteins were separated on 12% mini-SDS-PAGE with 6 M urea and blotted 1h to PVDF using semi-dry transfer (Hoefer). Membranes were blocked with 5 % milk 0,05% TBS-T for 1h at room temperature (RT) with slow agitation and after a brief rinsing with TBS-T they were incubated in the primary antibody at a dilution of 1: 1000 1% milk TBS-T overnight at 4°C with slow agitation. The antibody solution was decanted and the blot was rinsed briefly twice and washed once for 10 min with fast agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:25 000 in 1 % milk TBS-T for 1h at RT with slow agitation. The blot was washed as above and developed for 5 min with ECL Western Blotting Detection Reagent. Exposure time with Fuji X-ray films for optimal developing was 3 min.
Courtesy of Maija Lespinasse, University of Turku, Finland
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