SUMO3 | Small ubiquitin-like modifier protein 3 (peptide antibody)
AS08 349 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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Protein, tagged with a 6xhis-tag, was expressed from a strain of E. coli that reconstituted the sumoylation system with SUMO3 (described in Elrouby et al). Cells were harvested and lysated by sonication in buffer containing 6M urea. Proteins were used to load a Nickel column under denaturing conditions with buffers containing 6M urea. A pH gradient was applied to release the specifically bound proteins and 0.5 ml aliquots were collected. The presence of the desired protein in each aliquot was checked in SDS-PAGE by Comassie staining. 3 ul of the aliquot showing the most intense signal were separated on 10% SDS-PAGE and blotted 1h to PVDF using tank transfer. Blots were blocked with TBS-T (0.1%) and 5% dry-milk powder for 4 h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5000 overnight at 4ºC with agitation. The antibody solution was decanted and the blot was rinsed 4 times for 15 min in TBS-T (0.25%) at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602 from Agrisera ) diluted to 1:50 000 in TBS-T (0.1%) and 5% dry-milk powder for 1h at RT with agitation. The blot was washed as above and developed for 5 min with chemiluminescent detection reagent, according to the manufacturer's instructions. Exposure time was 15 second.
Courtesy of Dr. Concepción Almoguera, CSIC, Spain
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