Simple Western Frequently Asked Questions (FAQ)

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.

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.

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.

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.

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.

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.

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.

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.

• Stellar NIR/IR modules provide multiplexing with industry-leading fluorescence sensitivity and industry-lead detection sensitivity.
• With Superplex assays on Jess, you can pair 3-channel detection with simultaneous total protein normalization.
• RePlex on Jess and Abby performs sequential immunoassays, eliminating the traditional strip and reprobe of traditional Western blot.
• Instruments like Peggy Sue, Sally Sue, and NanoPro 1000 perform up to 8 cycles, allowing you to interrogate the same 5 µL of sample with 8 different antibodies.
• See our multiplex assay compatibility chart to find the right instrument for your assay.