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The Single-Cell Western™ protocol is highly versatile and can easily be adapted for different biological applications and protein targets. Researchers studying signaling proteins or other targets that require treatment of cells prior to analysis can add a drug, cytokine, or other form of pre-treatment directly to scWest chips after cells have been captured and before running the chip on Milo. Cells can also be treated just prior to settling them onto scWest chips. As a result, the flexible Single-Cell Western workflow allows time-dependent experimental manipulations to be easily performed before lysis and electrophoresis.
Here we describe a two-step, on-chip pre-treatment protocol to measure heterogeneity of modified histones — a class of proteins that is challenging to measure but critical in regulating gene expression. This new measurement capability could be key to unlocking new discoveries in the field of epigenetics.
Every cell is unique, making cell-to-cell heterogeneity important in many areas of biomedicine including cancer pathogenesis, immuno-oncology and regenerative medicine. More and more high-profile publications are using single-cell analysis techniques to reveal variability in cellular response to a drug or stimulus. They're also uncovering variation in drug target expression within a tissue and identifying important subpopulations of cells within complex samples that play key roles in disease progression. Single-cell protein expression information is critical when you need to understand the fundamental composition and behavior of complex biological samples.
Milo, the first and only Single-Cell Western platform out there, lets you do Western blotting at the single-cell level. Now you can run Westerns on thousands of individual cells in parallel and get robust, Western-based information on protein expression heterogeneity in your cells. He also multiplexes so you can measure multiple proteins in each single cell. That means you can get a better understanding of correlations between target expression and characterize cell signaling in specific target-positive subpopulations of cells. Where your target is located in a cell doesn't matter to Milo — his fast, simple workflow lets you measure proteins both on and in each individual cell with the same workflow. It's easy to detect surface proteins and you don't have to worry about fixing and permeabilizing your sample to measure intracellular proteins. As an added bonus, Milo uses conventional Western antibodies which means you can measure diverse protein targets — even ones that don't have good flow cytometry antibodies. The best part? Scout™ Software automates your data analysis and gives you quantitative protein expression measurements in each single cell.
Milo enables scientists to perform single-cell resolution Westerns (scWesterns) for over 1,000 individual cells simultaneously, and in a fraction of the time of conventional Westerns. Researchers can now gain selective protein expression information for up to four protein targets in each cell, offering views into cell-to-cell variation within a complex sample.