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Superplex Your Western Blots

Assay in chemiluminescence, 2-color fluorescence, total protein, and protein normalization at the same time.

What is Superplexing?

Jess' "Superplexing" goes so far beyond what's ever been done before with multiplexing on Western blot and other protein analysis systems, we just had to call it something else.

Western blots are the standard laboratory method for detecting and analyzing total proteins and protein normalization. They are based on a proven methodology with extremely high accuracy and are a fundamental technique used in labs and research studies globally. Here at ProteinSimple, we are taking Western blots to the next level with instruments which conduct multiple tests for targets and parameters simultaneously.

Superplexing lets you do everything you've always wanted to:

  • Run multiplexed chemiluminescence and fluorescence assays 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
  • Do chemi and fluorescent multiplexing assays in parallel just to be absolutely sure you didn't miss a protein
  • 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

Superplex Western Blots with Jess

Only on Jess.

Superplex Western Assay Capabilities

Multiplex up to three channels to detect protein targets

When you Superplex with Jess, you've got one chemiluminescence and two-color fluorescence (NIR and IR) channels to detect protein targets in your Western blot tests. There's no one way to go—just mix and match to fit your needs for a specific sample or project. And you'll never have to give one up to run total protein because that happens on a totally different channel.

3 Channel Multiplexing Western Blot

Superplexed Simple Western Assay Principle. Your sample, separation matrix, stacking matrix, antibodies and reagents are loaded automatically, and all steps of a Superplexed immunoassay take place in a single capillary. Jess first detects the total amount of protein present in your sample and then probes for your targets of interest in order of fluorescence-based detection first, followed by chemiluminescence.

Simple Western size assays give you all the flexibility you need to detect multiple protein targets using Western blot testing. Use ultra-sensitive chemi assays for targets like phosphorylated proteins. When you want to do traditional multiplexing to look at two proteins with the same molecular weight (MW), just use NIR and IR fluorescence to see both. Got a third target at the same MW? Do a 3-plex and spot that one with chemi while you're looking at the other two with fluorescence. You can also look at multiple targets with different molecular weights anytime you need to. Plan on publishing your data or just want a little extra confidence in your results? Just have Jess run a protein normalization assay for you at the same time, don't even bother with HKPs. And when you need to kick that article out fast, Superplexing on Jess clocks in with at least 27 data points/hr with protein normalization on 25 samples within 3.5 hours.

Multiplex Western Blotting Pros and Cons

Use Superplexing for better results and fewer drawbacks

Until now, multiplexing Western blots was the only way to go—but we all know the roadblocks that come with it. Superplexing lets you keep everything you like about multiplexing now and eliminate what you don't.

Click to enlarge animation


The Superplex Workflow and Data on Jess

Getting more Western blot data points in less time

When you Superplex with Jess, one tiny 3 µL 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.

Setting up the Superplex test on Jess is simple. Load samples, antibodies and reagents into a plate. Then load the plate a capillary cartridge into the Jess instrument. She takes care of everything from there. Compass for Simple Western software automatically analyzes the data when she's done. That includes normalizing target data, so no need to bust out the pencil and TI-86.

Western blot results quantitative protein expression: Jurkat cells treated with Calyculin A show increased Akt phosphorylation at S473

Jurkat cells treated with Calyculin A show increased Akt phosphorylation at S473. Jurkat cells treated with Calyculin A show increased Akt phosphorylation at S473. Lane view of Akt and p-Akt detection in Compass for Simple Western software using the NIR (red) and chemiluminescence (black) channels, respectively (A). Graph view of e-gram overlays in Compass showing p-Akt (chemi) and Akt (NIR) protein peaks using 0.6 mg/mL of Jurkat whole cell lysate (blue and green trace) and Jurkat + Calyculin A lysate (orange and fuchsia trace) (B). Peaks Table in Compass for Simple Western software showing quantitative target protein expression data for each capillary (C).

Need to crunch the numbers for external normalization or to plot target protein expression against the total amount of protein in each capillary? No problem. Compass lets you export your data in a format that’ll let you do that.

Using Superplexing to analyze normalized protein data: Results show comparable p-Akt signals for increasing concentrations of Jurkat + Calyculin A lysate

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 (A) and total Akt detected by the NIR channel (B) as a measure of raw peak area (orange bars) and separate-channel normalization for the normalized peak area (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.

Stop multiplexing. Start Superplexing.

Western blotting on the Jess Instrument