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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. While all Simple Western users can multiplex proteins that have disparate molecular weights in the same chemiluminescence channel by utilizing the SDS-PAGE separation which separates proteins by size before immunodetection, several additional multiplexing options are available to Simple Western users of instruments like Jess™ and Abby™. These additional multiplex methods enable simultaneous detection of multiple proteins, even with overlapping molecular weights. With Simple Western, you get rich and quantitative multiplexed protein characterization data with just 3 µL of sample, and it’s fully automated so you can design your next experiment while Simple Western does the work for you.

Multiplexing Assay Strategies

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:

Simple Western Multiplexing Strategies Image

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.

Multiplexing graphs Simple Western

Multiplexing proteins using primary antibodies from different host species. 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.

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.

Superplex Capillary on Simple Western

With Jess, you can combine size- and channel-based multiplexing for even more flexibility called Superplexing. Jess' Superplexing goes far beyond what's ever been done before with multiplexing on Western blot and other protein analysis systems. Assay in chemiluminescence, 2-color fluorescence, total protein, and protein normalization at the same time.

Serial Multiplexing: Get Even More Data Per Sample With Replex

The strip-and-reprobe technique in traditional Western blot is widely used to gain more information from a single blot, thus saving on precious or costly samples. However, this technique is well known to suffer from unpredictable protein loss and typically requires lengthy optimization to achieve optimal stripping efficiency and maximize protein retention on the blot. This makes any quantitative approach using standard strip and reprobe techniques nearly impossible. RePlex™ on Simple Western replaces traditional stripping and reprobing to enable high fidelity serial rounds of immunoprobing. 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! Pair this with size-based or detection channel-based multiplexing strategies and you will be swimming in data.

A RePlex Simple Western Assay combines the time-saving benefits of an automated immunoassay with multi-target detection and protein normalization in the same capillary, resulting in more data and quantitation with less sample With RePlex, you can compare a chemiluminescent immunoassay with another, a chemiluminescent probe with a Total Protein assay or probe using chemiluminescence and then reprobe with NIR. The possibilities are endless!

Replex Diagram for Simple Western