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PLA and Single Cell Proteomics
National facility
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The facility employs proximity-based assays for high-specificity protein analyses. Antibodies conjugated to DNA oligonucleotides are used as affinity reagents to allow very sensitive protein detection with a high capacity for multiplexing.
SERVICES
- In situ proximity ligation assays (PLA) in fixed cells or tissue sections to image, with enhanced specificity, the presence of specific proteins, interactions and/or post-translational modifications to e.g. characterise acts of protein signalling in tumor tissues, or to evaluate effects of drug treatment.
- PLA to detect surface proteins of cells or extracellular vesicles using flow cytometric readout.
- PLA to increase sensitivity and specificity of Western blot experiments.
- Other proximity based single- or multiplex assays with dual or triple recognition of the target protein for highly sensitive and specific protein detection in solutions such as plasma and cell/tissue lysate.
- Conjugation of oligonucleotides to antibodies for custom single-binder or proximity-based protein assays.
- Access to state-of-the art methods to detect proteins, or proteins combined with mRNA transcripts, in single cells.
APPLICATIONS
- Visualise and quantify proteins, their interactions and post-translational modifications in many types of samples including fixed cells and tissues, cell and tissue lysates, and plasma.
- Analyse biological pathways and cell signalling.
- Study extracellular vesicles based on protein content or surface proteins.
- Investigate heterogeneity in cell populations or tissues at the level of protein (possibly in combination with mRNA).
- Validate or follow-up findings from transcriptomic studies and investigate RNA-protein expression dynamics.
- Quantitatively analyse of the effect of drug candidates on protein expression, interactions and modifications.
EQUIPMENT
- Microscopes:
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epifluorescence
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confocal
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automated scanning
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- C1 Single-Cell Auto Prep system, Fluidigm
- Realtime qPCR instrument for standard 96- and 384-well plates
- Access to BioMark HD system, Fluidigm
- Plate reader for absorbance and fluorescence detection
RECENT PROJECTS
- Löf L et al. Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout. Sci rep. 2016 Sep 29;6:34358
- Genshaft AS et al. Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction. Genome Biol. 2016 Sep 19;17(1):188
- Darmanis D, Gallant CJ et al. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells. Cell Rep. 2016 Jan 12;14(2):380-9
