Simple Western™
Simple Western capillary immunoassay platforms provide high-resolution protein separation and characterization with fully quantitative protein expression analysis in complex biological samples. Simple Western assays take place inside an automated benchtop instrument that seamlessly combines capillary electrophoresis (CE) with high-sensitivity immunoassay and total protein measurements directly in the capillary. Simple Western is the only fully automated Western blot on the market and offers superior analytical quantitation, sensitivity, and reproducibility. It’s like Western blot meets ELISA in one.
Simple Western is an open immunoassay platform, and any antibody may be used to develop custom assays for detection of target proteins and/or target protein isoforms with flexible multiplex strategies.
Find Bethyl antibodies validated for use in Simple Western
How Does Simple Western Work?
Simple Western is the only platform on the market that provides a fully automated Western blotting workflow, offering high reproducibility, sensitivity, and quantitation, in addition to time and cost savings. The manual steps of traditional Western blot are precisely controlled inside an automated capillary benchtop instrument. Simple Western uses CE-SDS to separate protein by molecular weight (SW-Size) or by isoelectric point using capillary isoelectric focusing (SW-Charge).
Once protein separation is complete, proteins are covalently immobilized to the capillary wall, and the steps of traditional Western blot like washing, antibody incubation, and immunodetection are performed without user intervention. Upon completion, analyzed results automatically appear in the Compass for Simple Western software and quantitative data may be exported for further analysis.
Why Use Simple Western?
As a capillary-based immunoassay, Simple Western automates the steps of traditional Western blots inside of a precisely controlled capillary environment and eliminates unreliable gel-to-membrane transfers. While Western blots are primarily qualitative and poorly reproducible, Simple Western produces fully quantitative and highly reproducible data with up to 6-log dynamic range and CVs consistently below 15%. Compared to Western blots, Simple Western requires only a fraction of the sample needed for analysis, as little as 3 µL.
Simple Western’s Jess instrument provides flexible multiplex detection strategies in chemiluminescence and fluorescence channels, and the Stellar™ NIR/IR Modules provide industry-leading detection sensitivity that is 100X more sensitive than traditional fluorescence Western blots. Simple Western provides simultaneous total protein detection, eliminating the use of unreliable housekeeping proteins for data normalization and allowing for more accurate protein expression measurements.
Simple Western’s RePlex™ feature performs sequential immunoassay or total protein detection in the same capillary, eliminating the strip and re-probe method of traditional Western blotting. Because samples are covalently immobilized to the capillary wall, RePlex completely and reproducibly removes antibodies between probing cycles. As a result, RePlex assays are not impacted by signal interference or sample loss between probing cycles.
Simple Western covalently immobilizes samples even within a complex matrix, so Simple Western reduces matrix effects that can occur with ELISA. Following immobilization, Simple Western flushes the sample matrix from the capillary before immunodetection in an optimized buffer environment, further reducing matrix effects.
Simple Western only needs one target-specific antibody to give specific detection, which simplifies assay development compared to custom sandwich ELISA methods. Protein separation profiles on Simple Western allow you to identify protein isoforms or off-target antibody cross-reactivity.
Simple Western Resources
View additional information and resources to understand best practices and applications of Simple Western experiments in action.
Simple Western FAQs
Results are ready in as little as 3 hours.
Jess and Abby process up to 25 samples per run. Peggy Sue, Sally Sue, and NanoPro 1000 process up to 96 samples per run.
As little as 3 µL of sample is required and as few as 25–30 cells can be analyzed per assay.
Compass for Simple Western is all you need to set up Simple Western assays and analyze results.
Simple Western is an open platform so any antibody may be used for detection, including western blot antibodies. Even antibodies validated for other applications such as FACS or IHC may work on Simple Western. Like other immunoassays, the primary antibody for your target should be tested and validated on Simple Western. View our list of Simple Western validated antibodies. Validated antibodies are continually added to our database and provide recommended dilutions saving time in assay development.
The observed molecular weight of a protein may be influenced by the protein standard used, the nature of the separation matrix, and post-translational modifications. Simple Western relies on CE-SDS and not SDS-PAGE to separate proteins. Therefore, an observed molecular weight on Simple Western may differ from the predicted molecular weight or the molecular weight observed on traditional SDS-PAGE, but this does not invalidate results.
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