Single Cell Protein Detection (Protein Simple Milo)
Protein Simple Milo: Single Cell Western Blots
The Milo single-cell Western blot platform measures the expression of multiple proteins across 1,000+ cells in just 4-6 hours. Simply provide your single-cell suspension and compatible primary antibodies. The AA Core will perform the services and return fluorescence images that can be used to quantify protein expression. The Core will assist with software analysis. Milo is a powerful tool both alone and in combination with the Core’s other single cell services: 10x Genomics, Fluidigm C1, and Fluidigm BioMark HD.
Applications of the Milo system include:
- scRNA-Seq validation
- Cell subtype, protein isoform, and phospho-protein heterogeneity identification
- Histone modification detection
- Gene editing efficiency determination
- Protein expression in low-abundance samples
Cell capture is completed by placing 1 mL of suspended cells (10,000-100,000 cells) over a single-cell Western (scWest) chip containing thousands of tiny microwells patterned into pre-cast, photoactive polyacrylamide gel. The Milo system then lyses cells, performs rapid SDS-PAGE separation (~1 min), and immobilizes proteins using UV light. No transfer to membranes is required. Loaded chips are probed with an antibody cocktail. Chips are then ready to be imaged. Importantly, the chips can be stripped and reprobed for additional proteins.
Protein Simple Milo
FACS: Sample preparation via FACS can be completed by the Core. Contact the Core Manager to arrange a consultation.
Cell Capture & Antibody Probing: Please provide a single-cell suspension containing >10,000 cells (10,000-100,000 cells/mL) and up to two Western-validated primary antibodies. Primary antibodies must be generated in different species for multiplexing. The Core provides all other reagents and supplies. Click here for a selection of Milo-tested antibodies.
Imaging & Analysis: The Core will provide scWest imaging service for the user. Fluorescence quantitation can be completed using Protein Simple’s free Scout Software. The Core will assist users with software usage.
|Service||Price*||UNC/NCSU/CGIBD Member Price**|
|Cell capture, Probing, & Imaging||~$700||~$450|
* Price estimate for standard service. A customized quote can be provided upon consultation.
** Investigators affiliated with UNC, NCSU, or the CGIBD receive a ~35% discount
How to start a project:
To begin a project using the Milo system, please contact the Core Manager to arrange a consultation.
Requirements & compatibility
Sample type: Suspension containing >10,000 cells (10,000-100,000 cells/mL)
Cell diameter: 7–25 μm
Cell type: Mammalian cells; in suspension and unfixed
Antibody requirement: Standard, commercial unlabeled primaries and fluorescent secondaries
Other equipment needed: Open-format fluorescence microarray scanner capable of 10 μm resolution
Performance & specifications
Typical capture & analysis yield: 1,000–2,000 cells per scWest chip
Molecular weight (MW) range: 15–175 kDa
Typical target multiplexing: Up to four proteins per cell based on spectral and size-based multiplexing, and antibody species compatibility. Currently, the AA core can detect up to 2 proteins per staining with the available 2-color scanner.