Single Cell Multiplexing with Milo

A Powerful Single Cell Proteomics Solution

Single Cell Multiplexing up to 12 Targets Per Cell

Single Cell Westerns with Milo™ allow you to unlock single cell proteomics and measure changes in protein expression heterogeneity that aren’t possible with other techniques. Whether you need to conserve limited samples, compare relative abundance of protein isoforms or a phosphorylated form to its total abundance, assess the expression of proteins with a similar molecular weight, or just want to confirm a result with a different antibody, Milo is a powerful solution that lets you do all of this at the single cell level. Make the most of your research with single cell multiplexing on Milo.

Learn How to Do Single Cell Multiplexing with Milo

Single cell multiplexing can provide you with the flexibility required to get the most data points out of each sample. Milo provides single cell multiplexing strategies via spectral-based or size-based detection capabilities, where one antibody cocktail can be stripped off and the chip reprobed with a new set of antibodies.

  • Spectral multiplexing: Use antibodies labeled with distinct fluorophores and image them in different spectral channels on your microarray scanner. For example, mouse and rabbit primary antibodies can be spectrally differentiated by using a donkey anti-mouse Cy3 and donkey anti-rabbit Cy5, respectively.
  • Size-based multiplexing: Use the SDS-PAGE separation to resolve proteins that differ in molecular weight. Probing and imaging can be done using primary antibodies from the same host species or secondary antibodies that fall in the same spectral channel.
  • Stripping & re-probing: Since your sample is covalently bound into the gel after the SDS-PAGE separation and UV light exposure, antibodies can be stripped off with minimal sample losses. This enables multiple rounds of stripping & reprobing on Milo – something that isn’t typically possible with conventional Westerns which rely on weaker electrostatic or hydrophobic interactions to immobilize the sample on a membrane.

Milo scWest chips permit 10-plex to 12-plex Single Cell Western assays using a basic two-color scanner — on par with the multiplexing capability of high-end flow cytometers. Using this stripping and reprobing strategy with three or four color scanners could yield even higher multiplexing levels. 

Our technical note will walk you through multiplexing with Milo and provide you with evidence of and guidance for efficient stripping to achieve multiplexing levels that match or exceed those of the most powerful multiplexed flow cytometers.