Run two immunoassays on the same capillary—you choose the probing order
Lane view in Compass for Simple Western shows similar band intensities and quantitation shows comparable peak areas for AKT1 and AKT2 in each probing cycle.
Signal intensity and reproducibility for AKT1 and AKT2 in MCF7 lysates, detected in Probe 1 or Probe 2 in a RePlex assay. Regardless of the order in which each protein was probed, AKT1 and AKT2 in MCF7 lysates detected in Probe 1 or Probe 2 in a RePlex assay show excellent reproducibility and similar signal intensity across both probing cycles.
Stripping and reprobing are a thing of the past—remove antibodies more efficiently using RePlex!
Antibody probes are efficiently removed during a RePlex assay. To determine removal efficiency, primary and secondary antibodies from Probe 1 were removed from the protein in the automated assay and then incubated with the same secondary antibody in Probe 2 to detect any residual primary antibody from Probe 1. Lane view shows immunoassay signal for Probe 1 and no residual signal for Probe 2 for multiple targets in Jurkat and MCF7 cell lines. Removal efficiency (%) for these targets was calculated as (Peak area Probe 1 – Peak area Probe 2)/Peak area Probe 1 x 100.
Automated immunoassays and total protein normalization equals better quantification
Immunoassay and total protein detection performed in a single capillary AKT phosphorylation in MCF7 lysates untreated and activated with h-IGF1. Phospho-AKT and pan AKT were detected in Probe 1 using chemiluminescence and NIR fluorescence, respectively, while total protein signal was detected in Probe 2 (A). Example Graph views of pan AKT, AKT Ser473 phosphorylation and total protein signal for samples in panel A (B). Peaks Table in Compass for Simple Western shows automated normalization of phosphorylated and pan AKT signal to total protein signal demonstrates quantitation of target protein expression. (C).
Get more data per sample using RePlex with Jess!
Talk to your sales representative about RePlexing with your Jess system today