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High Sensitivity Multiplexing and Total Protein Normalization

The understanding of signaling networks has significant potential to advance therapies in cancer and regenerative medicine. Simple Western™ from ProteinSimple, a Bio-Techne brand, is a capillary-based automated immunoassay that separates proteins by Charge or Size for specific and sensitive protein characterization using conventional Western blot antibodies.

  • Unlike the traditional Western blot, Simple Western assays generate reproducible and fully quantifiable data in as little as 3 hours. And it's all automated.
  • RePlex™ on Simple Western performs two sequential immunoassays in the same capillary, allowing users to quantify the relative phospho-isoforms to total protein isoforms of any target without the need to strip & reprobe.
  • Take multiplexing even further with chemiluminescence, infrared (IR), and near-infrared (NIR) detection channels.
  • Stop the use of housekeeping controls, which can fluctuate in response to cell stimuli with misleading results. Instead, use built-in Total Protein Detection for more accurate quantification of your phospho/pan protein pair in response to cell stimuli.
RePlex on Jess quantifies phospho and pan AKT expression with total protein normalization
Automated multiplex detection of phospho/pan AKT and total protein normalization with RePlex.
The automated high-throughput Simple Western assay can analyze up to 96 samples overnight, enabling the analysis of full signaling pathways in one run.

Analyze an Entire Signaling Pathway Overnight!

Have you ever wanted to analyze a full signaling cascade in each of your samples in a single experiment? Simple Western can analyze up to 96 samples in a single overnight run using only 5 μL of sample, which is ideal for gaining a comprehensive overview of many targets together with their phosphorylated isoforms in a complex signaling pathway in a single experiment. Follow our sample prep workflow for large sample numbers, then load your samples in Peggy Sue, Sally Sue, or NanoPro 1000 and let Simple Western take care of the rest!

Single-Cell Analysis

Single-Cell Westerns on Milo™ perform Western analysis on approximately 1000 individual cells to provide insight into population heterogeneity. Like Simple Western, Single-Cell Western uses conventional Western blot antibodies and is capable of resolving phosphorylated isoforms that are critical for understanding the activity of cell signaling networks. Taken together with Simple Western, these advanced Western blotting solutions offer protein characterization at both a population-level and single-cell resolution.

Milo performs Western analysis on individual cells to provide insight into population heterogeneity

Picking Up Signals: Simple Western and Single-Cell Western Map Cell Signaling

Simple Western and Single-Cell Western provide a holistic overview of signaling pathways in many different cell types, organisms, and environments, from the study of cancer to the impact of spaceflight.

Scientific Review on Simple Western and Single Cell Western in Cell Signaling

High Resolution PTM Isoform Analysis with Simple Western Charge

What is Simple Western Charge?

This capillary isoelectric focusing (cIEF) immunoassay has many advantages over slab gel IEF and Western blot.

Simple Western Charge Protocols

Detect and quantify nearly all protein isoforms with high sensitivity and resolution.

Detailed ERK1/2 Characterization

Resolve and quantify unphosphorylated, mono, and dual-phosphorylated isoforms of ERK1/2.

Protocol on Phosphatase Reaction for Simple Western Charge Assays

Phosphatase Reaction for Simple Western Charge

The phosphorylated peaks of a target protein in NanoPro™ 1000 and Peggy Sue™ Simple Western Charge assays can be identified by treating lysates with phosphatase and comparing the electropherograms to untreated control. Here, we provide a protocol that uses lambda phosphatase, a Mn2+-dependent protein phosphatase that releases phosphate groups from phosphorylated serine, threonine, tyrosine, and histidine residues.

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