Parallelism experiments were carried out to determine if the recombinant NBR1 standard accurately determines NBR1 concentrations in biological matrices. HeLa, 3T3 and C6 cells were lysed in RIPA Cell Lysis Buffer 2. Values were obtained using the cell lysates serially diluted in assay buffer and assessed from a standard curve using four parameter logistic curve fitting. The observed values were plotted against the dilution factors. Parallelism of the curves demonstrates that the antigen binding characteristics are similar enough to allow the accurate determination of native analyte levels in diluted samples from cell lines of Human, mouse and rat origin.
Human HeLa cells were treated with 800 nM Bafilomycin A1, an inhibitor of autophagy protein degradation, for 24 hours at 37°C. Treated and untreated cells were washed in PBS and lysed in RIPA Cell Lysis Buffer 2 with protease inhibitors. Treated and untreated lysate dilutions (1:2, 1:4, 1:8, 1:16, 1:32, and 1:64) were run on a 4-15% Tris-HCl gradient gel. Proteins were then transferred to a nitrocellulose membrane and probed with a monoclonal antibody against NBR1. The same lysates were also diluted in the assay buffer and run in this kit.
Representative Standard Curve using ab136944.