Single-Cell Western

Check out the products and resources from Novus Biologicals supporting single-cell western protein analysis and instrumentation.

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Validated Primary Antibodies

Single-Cell Western Consumables

Single-Cell Western FAQs

Milo User Guide

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Conventional western blots provide averaged protein expression information for thousands of pooled cells. These bulk measurements can miss biologically significant cell subpopulations within heterogeneous samples. However, with the Single-Cell Western application using novel technology from Milo, it is possible to examine single-cell protein expression with conventional western blot antibodies. Single-Cell Western users can measure variation of protein expression across heterogeneous cells within a sample. The Single-Cell Western application can provide protein expression of 1,000 cells per run with the ability to multiplex up to 4 antibodies per cell simultaneously. Moreover, with up to a 99% stripping efficiency and reprobing capability, up to 12 targets can be assessed per cell providing maximum protein expression information on a heterogenous set of cells. Milo is made by ProteinSimple, a Bio-Techne brand and leader in single-cell protein analysis technologies.

Experimental workflow of Single-Cell Westerns with data examples

What are the benefits of Single-Cell Westerns?

  • Use of standard western blot antibodies. Antibodies validated for Western blot can be used for Single-Cell Westerns, although they should be titrated for optimized performance on Milo. View validated primary antibodies.
  • Use less antibody. Typical primary antibody dilutions range from 1:5 to 1:20 and 1:10 to 1:40 for secondary antibodies (if indirect detection is performed). With a final working volume of 80 µL per chip, the amount of primary antibody required is less than that used in traditional Western Blot.
  • Gather more data on each cell. Single-cell resolution western blotting on Milo can simultaneously quantify phospho- and total protein expression in each individual cell, providing quantifiable data on phosphorylation state across stimulated and unstimulated cell populations.
  • Expand capabilities with multiplexing. To spectrally multiplex, probe your sample with a cocktail of primary antibodies raised in different host species. Using different spectrally tagged secondary antibodies, you can image your chip in multiple spectral channels. In addition, you can use differences in molecular weight of proteins to resolve them in the same spectral channel when targets have more than a 30% difference in molecular weight.
  • Run one experiment, get 12,000 results. Each chip provides data for approximately 1,000 individual cells per run in as little as 4 hours with minimal hands on time. With multiplexing capabilities, that number expands drastically. With the ability to perform multiple stripping and re-probing steps on the chips, up to 12 proteins per cell can be analyzed. Learn more about Stripping and Reprobing Your Single-Cell Westerns.
  • Maximize your research using rare samples. Proteins immobilized within the chip are stable and can re-probed up to 9 months later, allowing one to ask new questions using the same sample. With precious samples or time-intensive experimental conditions, Single-Cell Western analysis maximizes your research with numerous data points and possibilities for further analysis.
  • Complement your flow cytometry experiment. Milo can characterize highly enriched FACS-sorted cell populations which don't contain enough cells to analyze with other techniques. Single-Cell Westerns provide individual cell data for targets hard to detect in flow, often due to lack of antibody availability validated for flow cytometry. Learn how Milo can solve flow cytometry and FACS challenges.
  • Validate single-cell RNA-seq data with single-cell protein data. Scientists can split their sample and run half through single-cell RNAseq workflow and half through the single-cell western. Once RNA targets are determined, they then probe the scWest chips for their targets of interest and get direct protein expression validation of their single-cell RNA data on the same sample.

Multiplex and obtain more data on Milo by using primary antibodies of different hosts,  variable tagged secondary antibodies, or differences in molecular weight.

How do Single-Cell Westerns Work?

Single-Cell Westerns can be performed on any non-fixed single-cell solution, whether they be in suspension, adherent cells removed by trypsin, or dissociated cells from tissue. Load 1 mL of the cell suspension dropwise onto a scWest chip, which is a polyacrylamide gel lined glass chip with 6,400 microwells where proteins from individual cells can be separated by size on Milo. This semi-automated, simplified western blot technology allows you to generate single-cell protein expression data for 1,000 individual cells per run in as little as 4 hours with minimal hands on time.

Visualize Quantitate
Single-Cell Western allows visualization and quantitation for phospho and total proteins simultaneously. Single-Cell Western allows visualization and quantitation for phospho and total proteins simultaneously.

Using fluorescence-based scanners such as the 2-color InnoScan 710 or an equivalent scanner with 5 μm scanning resolution, image the scWest chips and analyze data with Scout software. Scout software is a powerful tool to automate and streamline your image analysis. Scout provides automated lane finding, peak detection, molecular weight sizing and signal quantification. With the ability to multiplex on each cell, Single-Cell Western analysis makes it possible to detect discrete single cell expression of multiple phospho- and total proteins in individual cells within a subpopulation of cells.

Single-Cell Western Antibodies

Novus Biologicals offers over 58,000 western blot validated primary antibodies and almost 1,000 secondary antibodies. Standard Western blot antibodies can be used for Single-Cell Western but require optimization on Milo. Primary antibodies can be tested in unvalidated species or new applications with our our Risk-Free Testing Program.

Choose from:

View all Western Blot-validated Primary Antibodies

Secondary Antibodies

HRP-conjugated secondary antibodies

FITC-conjugated secondary antibodies

Biotin-conjugated secondary antibodies

Custom Conjugation Service

Scout and Milo is a registered trademark of ProteinSimple.

Learn how to identify and characterize cell subpopulations with Single-Cell Westerns on Milo.