Simple Western Frequently Asked Questions (FAQ)

Simple Western Instruments - Jess & Peggy Sue Hero

What is a Simple Western instrument?

Simple Western™ instruments are benchtop automated protein analysis platforms that seamlessly combine capillary electrophoresis to separate proteins by size (CE-SDS) or charge (icIEF) with immunodetection.

Simple Western is the only fully automated and complete solution for protein detection and characterization that represents a true reinvention of Western blotting and traditional ELISAs. Simply load your sample, antibodies, and reagents, and insert the sample plate and cartridge into your Simple Western instrument. Come back to fully analyzed results in as little as 3 hours. 

Simple Western instruments include Jess™, Abby™, Peggy Sue™, Sally Sue™, and NanoPro™ 1000. 

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What does Simple Western test for?

Simple Western assays characterize target proteins with fully quantitative protein expression data. Simple Western performs immunoassays and total protein measurements for fully quantitative protein expression data and detailed characterization of protein biochemistry, including molecular weight, isoelectric point, and post-translational modifications like phospho-protein isoforms and glycosylation. 

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How much does a Simple Western instrument cost?

Request a quote or contact a sales specialist for pricing.

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Simple Western Size and Charge Separation Protein Assays Figure

How does Simple Western work?

Simple Western assays are automatically and precisely controlled inside a capillary at the nanoliter scale.

  • Proteins are separated by capillary electrophoresis, either by size (CE-SDS) or charge (icIEF).
  • Once separated, proteins are covalently immobilized to the capillary wall with proprietary UV-conjugating technology.
  • Simple Western automates washing, primary incubation, secondary antibody incubations, and finally detection, all performed seamlessly back-to-back.
  • Simple Western instruments and assay conditions are controlled with Compass for Simple Western software, which also performs data analysis after the run is complete.

Simple Western has flexible multiplex capabilities to detect multiple targets in chemiluminescence and IR/NIR fluorescence detection channels with best-in-class chemiluminescence and fluorescence detection sensitivity. Simple Western even perform simultaneous total protein normalization so you can normalize protein expression data with confidence.

What types of assays can I run on Simple Western instruments?

Simple Western combines CE-SDS or icIEF with immunodetection (like ELISA combined with protein separation). Size-based Simple Western assays separate proteins by molecular weight using CE-SDS from 4-440 kDa. Charge-based Simple Western assays separate proteins by charge using icIEF from pI gradient 3-10. Size-based Simple Western instruments also perform total protein assays and total protein normalization for accurate protein expression measurements.

What should I expect from a size-based assay with Simple Western?

To analyze proteins by size, samples and reagents are loaded into an assay plate and placed into instruments Jess, Wes, Sally Sue or Peggy Sue. Sample is loaded into the capillary automatically, electrophoresed and separated by size (CE-SDS) as they migrate through a stacking and separation matrix. The separated proteins are then immobilized to the capillary wall via a proprietary, photoactivated capture chemistry. Target proteins are identified using a primary antibody and immunoprobed using a conjugated secondary antibody. Sample data is displayed by lane in a virtual-blot like image similar to traditional Western blot results and an electropherogram view. The resulting chemiluminescent or fluorescent signals are detected and quantitated. Quantitative results such as molecular weight, signal intensity (area), percent area, and signal-to-noise for each immunodetected protein are presented in the results table automatically from Compass for Simple Western software.

Automated protein normalization Western blot data on Simple Western
Simple Western charge separation diagram

What should I expect from a charge-based assay with Simple Western?

Do you want to know how many site are phosphorylated and or glycosylated? Charge-based Simple Western assays can resolve all post-translational modifications of your target protein. To assess protein separation by charge, samples and reagents are loaded into an assay plate and placed into Peggy Sue or NanoPro 1000. Your sample loads into the capillary automatically and is separated by charge using icIEF to resolve proteins according to their isoelectric point (pI). The pI is the pH of a solution at which the net charge of a protein becomes zero. At solution pH that is above the pI, the surface of the protein is predominantly negatively charged, and therefore like-charged molecules will exhibit repulsive forces.

The separated proteins are then immobilized in the capillary via a proprietary, photoactivated capture chemistry. Target proteins are identified using a primary antibody and immunoprobed using an HRP-conjugated secondary antibody and chemiluminescent substrate. The resulting chemiluminescent signal is detected and quantitated. Your output will be electropherograms, which make it easier to see minute changes. Quantitative results such as pI value, signal intensity (area), percent area, and signal-to-noise for each immunodetected protein are presented in the results table automatically from Compass for Simple Western software.

How do I analyze Simple Western data?

Data generated by Simple Western instruments automatically appear in the Compass for Simple Western Software once the run has completed. Here, data may be further analyzed or exported directly for publication. This one-stop solution eliminates the need to pass between different pieces of equipment to analyze data, for instance the gel doc and computer and is amendable for uploading to appropriate data repositories or journals to improve transparency. If you’re working in Quality Control (QC) and Good Manufacturing Practice (GMP) environments and need to manage data integrity, Compass for Simple Western has integrated functionality for compliance with 21 CFR Part 11.

FDA 1 CFR Part 11 Compliant Tools

Simple Western data can be viewed in a variety of ways but is always reproducible and not subject to incorrect manipulations. Simple Western users may choose to view data in graph view, lane view, and they may even view the direct image of the capillary. In graph view, data is portrayed as an electropherogram, with size or charge-based migration distance on the X-axis and the signal intensity of the chemiluminescence or fluorescence readout on the Y-axis. This Cartesian representation of immunoassay data provides immediate quantitative information for signal intensity, which is derived from the area under the curve. For each peak in the electropherogram, Simple Western automatically calculates signal height, area, % area, width, S/N, and baseline values, and these numbers may easily be exported for further statistical analysis if desired.

Simple Western size-based electropherogram

Users of traditional Western blot may not be accustomed to viewing data portrayed as electropherograms. For this reason, Simple Western also offers a lane view feature that allows for data portrayal that more closely resembles the image of traditional Western blot. In this view, protein signal is not portrayed as in the Cartesian graph, but rather as virtually mimicked lanes on a gel. Compass for Simple Western eliminates the possibility of manipulating Western blot data that goes against data integrity guidelines. For example, the acceptable practice is that contrast and brightness of a gel image may be adjusted so long as it is applied to the entire blot. In accordance with this guideline, Compass for Simple Western allows users to adjust these parameters across all samples. Because each of the capillaries are independent, lanes can also be rearranged in the software, eliminating the need to rerun samples so that they are adjacent or manually splice lanes together, which is discouraged by journals.

 

Simple Western Compass Size Assay Lane View

What software do I need for Simple Western instruments?

Compass for Simple Western is all you need to set up Simple Western assay and analyze results. Compass for Simple Western will analyze Simple Western size or charge-based assay data and process the results for you. Data can be displayed in a lane view, graph view, or as a digital capillary image and can be easily shared. You may even use Compass for Simple Western to remotely access your results from home after setting up your Simple Western run in the lab. See our Remote Tools for Simple Western for more information. Compass for Simple Western is available for Windows and Mac.

 

How does Simple Western measure protein quantification?

Western blot data are primarily qualitative, and extracting quantitative information is difficult and often unreliable. With modern image analysis software, it is easy and therefore tempting to manipulate Western blot data and images, which can lead to a misrepresentation of the results. Simple Western overcomes the commonly encountered problems with traditional Western blot data by creating highly reproducible, quantitative protein data in one electronic data file which integrates experimental assay conditions, raw data and quantitative peak areas and other analyses. Unlike traditional Western blots, Simple Western is highly quantitative by nature, bypassing densitometry analysis altogether. See our White Paper on producing publication-ready results with Simple Western.

Jess Superplex Multiplex Immunoassay Compass Table

How is protein normalization performed on Simple Western?

A common use of Simple Western assays and other immunoassays is to quantify protein expression and measure changes in protein expression in response to cell stimuli or drug treatment. For this purpose, normalizing protein expression by a housekeeping protein can be misleading as many housekeeping proteins may change under conditions where their expression was previously assumed to be stable. This obfuscates protein expression results, and journals now strongly discourage the use of housekeeping or loading control proteins for protein normalization. Instead normalizing the expression of target protein to the overall protein abundance in the sample is a more accurate way to measure changes in expression.

Simple Western addresses these issues by offering total protein normalization that allows for direct comparison between runs. Normalizing data by total protein detection may be performed by the chemiluminescence-based total protein detection assay, which is compatible with all size-based Simple Western platforms, or by the fluorescence-based protein normalization on Jess.

Protein normalization lane view image

How long does it take to get Simple Western results?

Results are ready in as little as 3 hours on Jess and Abby, which process up to 25 samples per run. RePlex assays are approximately 5 hours. Peggy Sue, Sally Sue, and NanoPro 1000, which can process up to 96 samples per run, your results are ready overnight (10-18 hours).

How much sample do I need for Simple Western?

Jess and Abby require only 3 µL of sample loaded per well. For analysis with Peggy Sue, Sally Sue, and NanoPro 1000, 5 µL of sample is recommended and up to 8 data points can be generated from the same. As few as 25 cells are needed for Simple Western charge-based assays on Peggy Sue and NanoPro 1000.

simple western advantage
Jess vs Western Blotting Infographic

The difference between Simple Western and Western blot explained.

Simple Western automates all traditional Western blotting steps including protein separation, washing, antibody incubations, and detection inside a nanocapillary, thereby eliminating gels, blots, transfer devices, X-ray film, manual labor, and poor results. With Simple Western, all steps are precisely controlled and automated by a benchtop Simple Western instrument, like Jess, Abby, Peggy Sue, Sally Sue, or NanoPro 1000. During assay setup, the user has the option to change incubation times, sample and antibody combinations, and choose between immunoassay or total protein detection and flexible multiplex strategies like RePlex, Superplex, or Stellar. Like Western blot, Simple Western is an open platform, and any primary antibody may be used for the detection of target proteins. Unlike Western blot, Simple Western produces highly reproducible, fully quantitative results with a sensitivity that sets the industry standard.

What are the steps of running a Simple Western instrument vs Western blotting?

Simple Western requires only a short sample prep. Just load your samples and press start. Simple Western will take care of the rest. Automated liquid handlers like the CyBio Felix from Analytik Jena may be implemented to automate sample preparation and plate loading to streamline workflows even further. See the Application Note from Analytik Jena for more information.

How accurate is Simple Western vs Western blotting vs Western blot?

By eliminating the inherent variability of the gel-to-blot transfer and the manual steps of traditional Western blot, Simple Western is more reproducible than Western blot, with CVs consistent below 15%. Compared with traditional Western blot, Simple Western’s high-throughput capabilities facilitate running replicates and determining the linear range of detection. Simple Western can run 25 samples in 3 hours or 96 samples overnight. This makes it easy to run the appropriate number of replicates required to generate concrete data, to establish the linear range of detection, and validate antibodies. With chemiluminescence detection, the linear dynamic range can reach up to 6 logs depending on the antigen.

Jess vs Western Blot Header

My antibody is validated for Western blot. Does that mean it’s applicable for Simple Western?

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. Simple Western has a large and rapidly expanding database of validated antibodies. Check the Simple Western antibody database first to see if your antibody has already been validated.

How can I find a Simple Western-validated antibody?

Find your target antibody with the Simple Western antibody database! In addition to the Simple Western antibody database, Bio-Techne antibodies like those from Novus Biologicals and R&D Systems will indicate if the antibody has previously been validated on Simple Western. Even if you don't see Simple Western among the validated applications, the antibody may still work on Simple Western. As an open platform, antibodies from any vendor (not just Bio-Techne brands) may be used to detect your protein target! Even antibodies that detect DNA and lectins that detect glycans have been used on Simple Western.

Antibody validated in flow cytometry

What is the purpose of adding a secondary antibody?

A conjugated secondary antibody is used for detection. Secondary antibodies are conjugated to HRP for chemiluminescence detection or a fluorophore for NIR/IR fluorescence detection. Anti-rabbit, anti-mouse, anti-goat, and anti-human conjugated antibodies validated for Simple Western are available from ProteinSimple. Additional Simple Western-validated secondary antibodies including anti-sheep and anti-rat conjugated antibodies are available from R&D systems. Shop for secondary antibodies or build a custom Simple Western kit with our Kit Builder.

What should I do if my Simple Western validated antibody did not work?

Follow the Simple Western Assay Development Guide to be sure you used the right antibody concentration and sample type to validate your Simple Western antibody. If you are having trouble with your target of interest using a validated antibody, our technical team will be happy to help you investigate the issue and find a potential resolution. Contact us.

What antibody concentration should be used for Simple Western?

As with other immunoassays, every antibody should be titrated to suit your specific sample. Validated antibodies will likely give you a starting concentration. For antibody optimization/validation, it is recommended to start with 10 to 50-fold higher antibody concentration than what is used for traditional Western blot. While antibody concentrations may be higher on Simple Western, only 10 µL of antibody is needed to probe each sample. Find the recommended dilution for your target antibody with the Simple Western antibody database!

When should I use Simple Western vs Maurice?

Simple Western combines CE-SDS and icIEF separation with immunodetection to specifically detect target proteins even in complex sample types like whole-cell lysates and tissue homogenates. Simple Western can be used in upstream bioprocessing workflows with crude sample types as well as further downstream with pure or partially purified samples. Learn more about Simple Western Applications.

When should I use Simple Western vs ELISA?

While both Simple Western and ELISA are immunoassays capable of specifically detecting target proteins in complex sample types, Simple Western provides protein separation profiles by size (CE-SDS) or charge (icIEF). Simple Western has the advantage of reduced interference by matrix effects that can occur with ELISA analysis of challenging samples like brain tissue homogenates. With Simple Western, samples are covalently bound to the capillary wall, allowing for the thorough flushing of the sample matrix before analysis.

Where can I find Simple Western protocols?

Protocol on Phosphatase Reaction for Simple Western Charge Assays

Which Simple Western instrument is best used for my sample population?

Complex samples like tissue homogenates and whole-cell lysates as well as pure or partially purified samples may be analyzed on an instrument. Size-based Simple Western assays are performed on Jess, Abby, Peggy Sue, and Sally Sue. Jess offers the most flexibility for multiplex detection with chemiluminescence and two-color fluorescence channels. Stellar NIR/IR modules, which provide industry-leading fluorescence detection sensitivity, are only available on Jess. See our Multiplex Compatibility Chart.

For characterizing post-translational modifications like phospho-protein isoforms, the charge-based Peggy Sue or NanoPro 1000 instruments may provide the most detail. These systems automate all steps including sample loading, charge-based protein separation using icIEF, immunoprobing, detection and data analysis. Only requiring as few as 25 cells per assay, Peggy Sue or NanoPro 1000 can quantify low abundance proteins in limited cell populations in up to 96 samples at once.

Simple Western Instruments - Jess & Peggy Sue Hero

How many targets can I assess through Simple Western?

Simple Western on Jess takes multiplexing to a new level with flexible multi-channel detection in chemiluminescence and NIR/IR fluorescence channels, helping you identify and differentiate relative protein expression between samples in one shot.

Multiplexed chemiluminescence and fluorescence immunoassays plus total protein and protein normalization with automated Western blots via capillary electrophoresis.
Simple Western Multiplex Assays
The automated high-throughput Simple Western assay can analyze up to 96 samples overnight, enabling the analysis of full signaling pathways in one run.

Why do I observe a shift in the molecular weight of proteins?

An observed molecular weight molecular weight (MW) of a protein on a Western blot may differ from the observed molecular weight on Simple Western and does not invalidate the results. Like traditional Western blot assays, Simple Western measures observed MW, also known as apparent or relative MW.

When compared to the theoretical MW of a protein, the observed MW may be influenced by many factors such as the protein ladder or MW standard used, the nature of the separation matrix, and post-translational modifications. Because Simple Western relies on CE-SDS and not SDS-PAGE to separate protein, an observed MW of a protein on a Western blot may differ from the observed molecular weight on Simple Western and does not invalidate the results.

CE-SDS versus SDS-PAGE App note reviewing publications that site important factors to consider when comparing molecular weight (MW) data from CE-SDS and SDS-PAGE

What tissue homogenization buffer can be used for Simple Western?

Many buffers our compatible with Simple Western. A major advantage of Simple Western is that reducing agents like ß-mercaptoethanol do not interfere with results as they do on ELISA because the sample matrix is thoroughly flushed out before detection. See our Simple Western Buffer Compatibility Table.