Deg8 | protease Do-like 8 (chloroplastic)
AS14 2767 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Nicotiana tabacum
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47 | 38 kDa
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Total protein from Arabidopsis thaliana was extracted with buffer containing 0.2M tris-HCl pH 6.8, 10% Glycerol, 8% SDS, 20% β-mercaptoethanol, 5M Urea. 10 µg of chlorophyll were separated on 12 % SDS-PAGE using tank transfer and blotted 1.5h to NC. Blots were blocked with 5% nonfat dry milk in TBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 in 5% nonfat dry milk in TBS-T over night at 40C with agitation. The antibody solution was decanted and the blot was rinsed briefly once, then washed once for 5 min and 2 times for 15 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated from Sigma) diluted to 1:10000 in 5% nonfat dry milk in TBS-T for 1h at RT with agitation. The blot was washed as above and developed for 2 min with ECL according to the manufacturer instructions. Exposure time was 1 minute.
Courtesy of Leah Naveh, The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Israel
Deg8 protease forms a hexamer together with Deg5 in the thylakoid lumen and is involved in the cleavage of photodamaged D2 protein of photosystem II. Localized in thylakoid lumen.