PRP40B | pre-mRNA-processing protein 40B
AS14 2785 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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Total protein from rosette leaves, 30 ug of Arabidopsis thaliana whole leaf extract (prp40ab) (1), 30 ug of Arabidopsis thaliana whole leaf extract (wild-type) (2), extracted with buffer (100mM Tris, 10% glicerol, 5mM EDTA, 5mM EGTA, 0.15M NaCl, 0.75% Triton X100, 0.05% SDS, 1mM DTT) and denatured with 0.06M Tris-HCl, 5% glycerol, 2% SDS, 4% β-mercaptoethanol, 0.0025% bromophenol blue at 95 C for 5 min were separated on 8 % SDS-PAGE and blotted 1h to PVDF using semi-dry. Blots were blocked with 5% low-fat dried milk in TBS + 0,1%Tween for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 000 for 1h at RT with agitation in 2% low-fat dried milk in TBS + 0,1%Tween. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from ) diluted to 1:10 000 in 2% low-fat dried milk in TBS + 0,1%Tween for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL of high sensitivity. Exposure time was 60 seconds.
MW markers: PageRuler Plus Prestained (ThermoFisher)
Courtesy of Agata Stępień, Adam Mickiewicz University, Poland
PRP40B (pre-mRNA-processing protein 40B) protein that binds the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II and functions as a scaffold for RNA processing machineries. Ubiquitously expressed and localized to the nucleus.