Single-Cell Westerns

Milo™ is the world's first Single-Cell Western platform. He measures protein expression in thousands of single cells in a single run so you can profile heterogeneity in your samples. Just load your cell suspension and the scWest chip captures ~1,000 single-cells. Milo then does a fast, 1 minute SDS-PAGE separation on each single-cell lysate on-chip. Then just probe with your favorite conventional Western antibodies to measure ~12 proteins per cell using a variety of multiplexing strategies. Use Single-Cell Westerns to unlock the single-cell proteome and measure more of the proteome than is possible with any other single-cell technique.

Milo - Single-Cell Western - Simplify FACS Studies

How Can Milo Help You?

I need to profile heterogeneity in complex samples

Milo measures protein expression in ~1,000 single-cells in one 4 hour experiment so you can quantify expression heterogeneity of your target and the percentage of cells in your sample that are target positive.

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Milo chart - heterogeneity - 51% KRT8+ cells

I need protein validation of my single-cell RNA-seq data

Use conventional western antibodies to validate your single-cell RNA-seq data with single-cell protein data. Plus, scWest chips can be archived for up to 9 months after you run them so you have plenty of time to get your sequencing results back before you have to probe for your targets of interest.

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Milo chart - Protein validation of my single-cell RNA-seq data

I need multiplexing to detect multiple proteins at the single-cell level

Simultaneously detect 12+ proteins in your sample—all at the single-cell level—using spectral and size-based multiplexing strategies. You can even strip & reprobe scWest chips up to 9 times for higher multiplexed studies!

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Milo chart - multiplexing

I need to simplify my phospho-flow signaling studies

Eliminate the fixation and permeabilization steps of flow/FACS! Milo chemically lyses the cells captured on the scWest chip before analysis to gain access to intracellular and intranuclear compartments more easily than with flow cytometry. By lysing the cells, Milo can detect challenging proteins like transcription factors and even methylated histones!

You can also use Milo's sizing step to separate out non-specific binding, which is more common with phospho-specific antibodies and cannot be resolved with flow.

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Milo chart - STAT1 signaling - Eliminate the fixation and permeabilization steps of flow/FACS

I need to detect proteins that lack good flow antibodies

Milo is an open platform so you can use the large commercial catalog of Western antibodies which is 10-100x larger than the flow/FACS catalog. Don't get stuck without a flow antibody against your target of interest again.

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Milo chart - Milo allows you to use the large commercial catalog of Western antibodies which is 10-100x larger than the flow catalog.

I want to measure protein isoform heterogeneity

Milo's molecular weight sizing step can resolve protein isoforms that differ in molecular weight, allowing you to measure how many cells in your sample express one isoform, the other isoform, or both isoforms. Try that with flow!

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Milo chart - Measure protein isoform heterogeneity - 2 isoforms resolved by molecular weight

I need to measure gene editing efficiency or effect in low efficiency gene editing systems

Milo can quantify the percentage of cells in your sample that express an inserted gene, even in low efficiency systems. Plus, you can simultaneously measure your edited gene and downstream effect markers to understand the impact of the gene on the cells that have been edited.

Milo chart - Measure gene editing efficiency or effect in low efficiency gene editing systems

I need to measure protein expression in low abundance samples

Milo can analyze samples which contain as low as 10,000 single-cells so you no longer have to have collect millions of cells to analyze protein expression. The number of cells captured scales with the number of cells loaded so even lower abundance samples are possible.

Milo can also characterize highly enriched FACS-sorted cell populations which don't contain enough cells to analyze with other techniques.

Milo picture - Measure protein expression in low abundance samples and characterize highly enriched FACS-sorted cell populations

Application notes

Multiplexed Single-Cell Western Analysis of Red Blood Cells for Biomarker Detection in Blood and Neurodegenerative Disorders   
Going with The Flow: Using Milo to Streamline Your Flow Cytometry Experiments   
Generating Single-Cell Western Protein Heterogeneity Data with an InnoScan 710 Scanner   
[show details] 
Using Single-Cell Westerns to Validate Single-Cell RNA-Seq Data   
[show details] 
Reveal Cell Subtypes, Protein Isoforms, and Phospho-Protein Heterogeneity with Milo   
[show details] 
Adapting the Single-Cell Western Protocol to Detect Histone Modifications    
[show details] 
Protein Expression Heterogeneity with Milo, the First Single-Cell Western System   
[show details] 
The Single-Cell Western has Arrived   
[show details] 

Requirements & compatibility

Sample type Suspension containing >10,000 cells
Cell diameter 7-25 µm in suspension
Cell type Mammalian cells; globular in suspension and unfixed
Antibody requirement Standard unlabeled primaries and fluorescent secondaries
Other equipment needed Open-format fluorescence microarray scanner capable of 5 µm resolution

Performance & specifications

Typical cell dilutions Yield capture and analysis of 1,000-2,000 cells per scWest chip
Molecular weight (MW) range 15-175 kDa
MW resolution 10% differences in distinct spectral channels, as low as 30% differences in same spectral channel
Typical target multiplexing Up to four proteins per cell by spectral and size-based multiplexing
Twelve-plus proteins per cell using stripping & reprobing
Workflow time 4-6 hours