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Multiplex Protein Analysis with Simple Western

The capillary-based Simple Western™ platform is an established market leader in generating high-quality Western blot data and now is pushing the boundaries of multiplexing by enabling a variety of strategies for detection of multiple proteins in each sample. Do multiple immunoassays or combine your immunoassay data with total protein normalization using just 3 µL of sample. It’s fully automated so you can design your next experiment while Simple Western does the work for you.

Multiplex Strategies on Simple Western

Jess Instrument Simple Western

Molecular Weight Size-Based Multiplexing

Multiplexing based on molecular weight gives you the flexibility you need to detect multiple protein targets that vary in molecular weight within the same capillary. Get more out of your 3 µL sample! Multiplexing two targets in one capillary with Simple Western doubles the number of data points per sample and increases the data quantitation accuracy when you normalize to biological loading control or system control. Learn More about Molecular Weight Size-Based Multiplexing. Next time you multiplex a rabbit primary with an antibody raised in different species, use the new 20X Anti-Rabbit HRP Conjugate for the best results.

When you multiplex a rabbit primary with an antibody raised in different species, use the 20X Anti-Rabbit HRP Conjugate for the best results. A significant signal decrease is observed when the RTU anti-mouse and RTU anti-rabbit secondary antibodies were mixed 1:1 (left) for ERK1 (rabbit anti-ERK1 RTU, PN 042-206) and STAT3 (mouse anti-Stat3 CST, 9139, 1:50). Using the 20X anti-rabbit secondary mixed into the RTU anti-mouse secondary (right) resulted in no change of saturation and yields equivalent results to individually run samples.

Multiplexing graphs Simple Western

Detection Channel-Based Multiplexing

Detection channel-based multiplexing offers you the flexibility to analyze one sample in the same capillary using different detection channels. With Simple Western Assays, you have the ability to multiplex up to three channels to detect protein targets: chemiluminescence, NIR fluorescence and IR fluorescence. Or you can run three immunoassays using chemiluminescence, NIR, and IR fluorescence, plus use the Protein Normalization Assay Module to normalize your immunoassay data with total protein data in the same capillary. Setting up detection channel-based multiplexing is simple. First, select primary antibodies for your target(s) of interest from different host species. Then, just add samples, antibodies and reagents into a plate and load the plate and capillary cartridge into the instrument. Compass for Simple Western software automatically analyzes the data when the run is complete. That includes normalizing target data as well! Learn More about Multiplexed Fluorescent Westerns on Jess.

Stellar High Sensitivity Multiplex

  • Stellar IR/NIR Detection Modules set the industry standard for fluorescence Western bot detection sensitivity.
  • With Stellar, multiplex in two-color fluorescence channels with an immunoassay or with Stellar Total Protein Detection (DM-TP03) in the chemiluminescence channel.
  • The sensitivity of Stellar NIR/IR detection rivals that of chemiluminescence on Jess, and it is 100X more sensitive than traditional fluorescent Western blotting technology! 
Stellar Immunoassay and Total Protein Assay

Superplex up to 3 Immunoassays with Protein Normalization

  • Jess' Superplex assays goes far beyond what's ever been done before with multiplexing on Western blot and other protein analysis systems.
  • You can run three immunoassays using chemiluminescence, NIR, and IR fluorescence, and use the Protein Normalization Assay Module (DM-PN02) to normalize your immunoassay data with total protein data in the same capillary without giving up a detection channel.
Multiplexed chemiluminescence and fluorescence immunoassays plus total protein and protein normalization with automated Western blots via capillary electrophoresis.

Get Even More Data Per Sample with RePlex

  • With RePlex, you can run two serial immunoassays within the same capillary to get all your rich protein characterization data in a single detection channel!
  • Unlike the strip and re-probe method of traditional Western blots, there is no signal interference or sample loss between probing cycle because the sample is covalently bound to the capillary wall, allowing for a complete strip of the antibodies. 
  • The second cycle of RePlex may also be used to Total Protein Normalization (TPN) to normalize your immunoassay data in the first cycle. 
  • Pair this with size-based or detection channel-based multiplexing strategies and you will be swimming in data.
Second cycle of RePlex dedicated to total protein normalization

Simple Western is a powerful tool that enables a variety of multiplexing strategies to get multiplexed western blotting or multiplexed ELISA-like data. Do multiple immunoassays or combine your immunoassay data with total protein normalization – all on the same sample. Simple Western multiplexing strategies include: