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Superplex - More than a Multiplex Immunoassay

Superplex assays on Jess™ go far beyond the traditional multiplex immunoassay on Western blot and other protein analysis systems, so we named it something else. Jess automates the protein separation and immunodetection of traditional Western blotting, eliminating tedious and error-prone steps. With both multiplex fluorescent immunoassay and chemiluminescent detection capabilities combined with in-capillary protein normalization, you can efficiently develop an assay to get more data points out of one sample.

      Characterize post transduction protein expression using automated Simple Western machine Jess

      What is Superplex?

      Multiplex Up To Three Channels

      Superplexing using Jess’s infrared (IR), near-infrared (NIR) and chemiluminescence channels helps you identify and differentiate relative protein expression between samples while built-in Protein Normalization gives you the confidence you need in your results all in one shot. Your sample, antibodies, and Protein Normalization and other reagents are loaded automatically, and all steps of a Superplex multiplex immunoassay take place in a single capillary. Jess first detects the total amount of protein present in your sample with the Protein Normalization Module, and then probes for your targets of interest in order of multiplex fluorescent immunoassay first, followed by chemiluminescence detection.

      Multiplexed chemiluminescence and fluorescence immunoassays plus total protein and protein normalization with automated Western blots via capillary electrophoresis.
      (PN) Protein Normalization, (Chemi) Chemiluminescence, (FL NIR) Fluorescence in the Near-Infrared
      • Run chemiluminescence and fluorescence multiplex immunoassays on the same sample at the same time
      • Look at multiple targets and parameters simultaneously
      • Get info on protein abundance, isoform and size all at once
      • Throw in total protein without giving up a detection channel
      • Detect high and low abundance proteins in the same sample, even when they have identical molecular weights
      • Save time—your multiplexed, normalized, quantitative, size-based data will be ready in 3.5 hours
      • Conserve precious samples—Jess only needs 3 µL/sample
      Jess Superplex Multiplex Immunoassay Chemiluminescence Fluorescence Graph

      Superplexing is Simple

      All you have to do is load your samples, antibodies and reagents into a plate, insert the plate and capillary cartridge and press Start! Then enjoy 3 hours of handsfree run time to focus on your science, product development, or project deadline. When Jess is done, come back to fully analyzed and quantitative results! Compass for Simple Western automatically analyzes the data when the run is complete, including normalizing target data.

      Jess Superplex Multiplex Immunoassay Compass Table

      More Western Blot Data in Less Time

      When you Superplex with Jess, a multiplex immunoassay platform, just 3 µL of sample gives you at least 4 data points. Not to mention the time you save with your Western blots by doing four things at once, and that it only takes 3.5 hours to do them. But those aren't the only pros of Superplexing. Because each of those data points gets captured in the same capillary, you're not spending time comparing, infering and interpreting data across runs, samples, or lanes either. That means you get to make more informed, confident conclusions about your Western blot test results.

      Figure caption: Protein normalization reveals comparable p-Akt signals across increasing concentrations of Jurkat + Calyculin A lysate. Comparative data showing p-Akt protein expression detected on Jess’s chemiluminescence channel and total Akt detected by the NIR channel as a measure of raw peak area shown in orange bars and separate-channel normalization for the normalized peak area shown in blue bars. As expected, normalization of the raw data results in similar peak area across all three lysate concentrations. Values plotted are mean protein peak areas for samples run in triplicate.


      Jess Superplex Multiplex Immunoassay Graph Average Peak Area

      Simple Western Multiplex Immunoassay Compatibility

      Simple Western­ has several assay types for multiplex analysis. View the comparison of multiplex immunoassay platforms in the table below. You can also build your own assay using the Simple Western Kit Builder.

      Assay Type



      Separation Module

      Detection Modules


      Size-Based Multiplex Peggy Sue, Sally Sue • Chemi • Separation Modules for Peggy Sue or Sally Sue • Chemi Detection Modules or Total Protein Detection Module (DM-TP01) No

      Size-Based Multiplex


      • Chemi

      • Std Separation Modules

      • Fluor Separation Modules

      • Chemi Detection Modules or Total Protein Detection Module (DM-TP01)


      Channel-Based Multiplex


      • Chemi

      • Fluor

      • Fluor Separation Modules

      • Chemi Detection Modules or Total Protein Detection Module (DM-TP01)

      • NIR / IR Detection Modules




      • Chemi

      • Fluor

      • Fluor Separation Modules

      • Protein Normalization Module (DM-PN02)

      • Chemi Detection Modules

      • NIR / IR Detection Modules




      • Chemi

      • Fluor

      • Fluor Separation Modules

      • Chemi Detection Modules or Stellar Total Protein Detection Module (DM-TP03)

      • Stellar NIR / IR Detection Modules

      • Separation Modules for Peggy Sue and Sally Sue include all MW ranges (2-40, 12-230, 66-440 kDa)
      • Separation Modules for Jess and Abby include all MW ranges (2-40, 12-230, 66-440 kDa) and capillary cartridge formats (2 x and 8 x of 13 and 25 capillary cartridges)
      • NIR/IR Detection Modules include Anti-Mouse and Anti-Rabbit Modules
      • Chemi Detection Modules include Anti-Mouse, Anti-Rabbit, Anti-Goat, Anti-Human, Biotin Modules, and No-Secondary Detection Modules

      Multiplex Western Blotting vs Simple Western

      Simple Western is the ultimate multiplex Western blotting platform. Keep what you like about multiplex Western blots and eliminate what you don't.

      Western Blot

      Simple Western

      Cannot multiplex across chemiluminescence and fluorescence channels Multiplex across chemiluminescence and NIR/IR fluorescence channels with simultaneous total protein detection
      Tedious and heavily hands-on traditional Western blot workflow Fully automated Western analysis following a simple sample preparation
      Challenging to detect low abundance proteins Industry-leading fluorescence detection sensitivity with Stellar NIR/IR Modules
      Gel-to-membrane transfer variability No gel-to-membrane transfer variability, highly reproducible
      Up to 50 µL sample required 3 µL sample required
      Semi-quantitative Fully quantitative
      >12 hours to results 3 hours to results (5 hours with RePlex)
      No single-step total protein detection Automated total protein normalization with multiple total protein detection methods
      Laborious strip and re-probe procedure often results in signal loss and/or interference RePlex automates sequential Western analysis with no signal loss or interference because samples are covalently bound to the capillary
      Higher cost per run compared to Simple Western Lower cost per run compared to Western blot
      Challenging to resolve high MW proteins Resolve and quantify high MW proteins like Dystrophin