Multiplex Assays on Simple Western

There are several techniques that can be used to gain protein expression data from multiple protein targets from small sample volumes using Simple Western Technology. Simple Western assays allow for more efficient use of small sample volumes (e.g., 3 µL) compared to traditional western blotting, enabling simultaneous analysis of multiple protein targets, isoforms, and sizes. These method described below provide quantitative results with minimal hands-on time, using both fluorescent and chemiluminescent detection to maximize data points from a single sample, including protein normalization.

Multi-channel Detection | RePlex: Automated Strip and Reprobe | Resampling | Multiplex with Total Protein Normalization

Multi-channel Detection

Simple Western gives you high quality, size-based data in just three short hours with only one hour of hands-on time. Multiplex in one capillary with sensitive chemiluminescence and fluorescence NIR/IR channels and detect up to 8 targets per sample.

RePlex detection of concomitant phosphorylation for AKT and downstream targets in the same capillary. (A) Lane view of AKT, GSK3b and cRaf phosphorylation in control (Ctl) and LY294002-treated (LY) Jurkat lysates. (B) RePlex enables use of the same secondary antibody in Probe 1 and Probe 2, with no carryover signal, as demonstrated by detection of phosphorylated AKT and GSK3b using rabbit-derived primary antibodies in sequential probes of the same sample. (C) Quantitation shows a decrease in GSK3b and cRaf phosphorylation in LY294002-treated Jurkat lysates. Error bars represent standard deviations of the means.

Resample Up to Four Times

Simple Western Leo allows for the automatic re-sampling or use of the same sample for multiple runs, which enhances experimental efficiency. You can set up experiment to measure the expression or levels of different target proteins from a single sample by using different capillaries. Leo, the largest Simple Western Instrument utilizes 25-100 capillaries to run 25-100 samples per run enabling the detection of up to 24 protein targets per sample. Each sample can be re-sampled up to 4 times and analyzed in separate capillaries. This ability to perform multiple analyses from the same sample reduces sample consumption and minimizes the need for repetitive sample preparation, while still providing accurate, quantitative data for various protein targets in a streamlined and automated process.

A single row of samples was loaded onto the sample plate, consisting of eight replicates each of HeLa lysates at 0.3, 0.2, and 0.1 mg/mL, for a total of 24 samples

Multiplex with Total Protein Normalization

With Simple Western Technology, reproducible total protein normalization (TPN) is integrated seamlessly with your immunoassay. Choose from one of two TPN assay strategies:

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