Recombinant, full-length RPN12a of Arabidopsis thaliana protein sequence UniProt: Q9SGW3-1, TAIR: At1g64520, overexpressed in E.coli
Certain proportion of both wild-type transcript and protein is still produced in the rpn12a -1 mutant, see Figures 2B and 2C Smalle et al. (2002), PVDF membrane instead of nitrocellulose, and use the primary antibody at a 1:3000 dilution in 1% non-fat dr
Certain proportion of both wild-type transcript and protein is still produced in the rpn12a -1 mutant, see Figures 2B and 2C Smalle et al. (2002), PVDF membrane instead of nitrocellulose, and use the primary antibody at a 1:3000 dilution in 1% non-fat dry milk in PBS, performing just a 1 hour incubation at room temperature, so those adjustments to the protocol may also help reduce the background and increase signal intensity. Recommended Western blot conditions: protein load: 10-20 µg/well, membrane: PVDF membrane, blocking with 10% nonfat milk 1h/RT, antibody incubation buffer: PBS-T. Primary antibody dilution: 1: 3000 in PBS with non-fat dry milk incubation RT/1h, optimisation based on obtained result regarding signal/noise ratio.
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