Multiplex Assay Strategies 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 Assay Strategies with Simple Western

Learn About Simple Western
Jess Instrument Simple Western

Size-Based Multiplex Assay

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. 

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.

Learn About Size-Based Multiplex
Multiplexing graphs Simple Western

Channel-Based Multiplex Assay

Detection channel-based multiplexing on Jess offers you the flexibility to analyze one sample in the same capillary. You have the ability to multiplex up to three channels to detect protein targets: chemiluminescence, NIR fluorescence and IR fluorescence. There is an extra channel for Protein Normalization so you can normalize your multiplex immunoassay data by total protein.

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!
Learn About Stellar
Stellar Fluorescence 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.
Learn About Superplex
Multiplexed chemiluminescence and fluorescence immunoassays plus total protein and protein normalization with automated Western blots via capillary electrophoresis.

Serial Multiplex Assay with RePlex

Never strip and re-probe again! RePlex™ on Jess and Abby provides high fidelity serial rounds of immunoprobing by removing the antibodies from the first probing cycle for a second probing cycle with fresh antibodies or Total Protein Detection.

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.
Learn About RePlex
Second cycle of RePlex dedicated to total protein normalization

Simple Western Multiplex Assay Compatibility Chart

Simple Western­ has several multiplex assays for protein analysis. See how they are compatible in the table below. You can also build your own assay using the Simple Western Kit Builder.

Assay Type

Instrument

Detection

Separation Module

Detection Modules

RePlex
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

 Jess, Abby

• Chemi

• Std Separation Modules

• Fluor Separation Modules

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

 Yes

Channel-Based Multiplex

 Jess

• Chemi

• Fluor

• Fluor Separation Modules

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

• NIR / IR Detection Modules

 Yes

Superplex

 Jess

• Chemi

• Fluor

• Fluor Separation Modules

• Protein Normalization Module (DM-PN02)

• Chemi Detection Modules

• NIR / IR Detection Modules

 No

Stellar

 Jess

• Chemi

• Fluor

• Fluor Separation Modules

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

• Stellar NIR / IR Detection Modules

 No
  • 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