Multiplexing: Faster Answers, New ChallengesMultiplexing, using the Luminex xMAP platform, provides faster answers to these cell signaling questions compared to traditional Western blots, mass spectrometry analyses and radioactive phosphorylation assays requiring large amounts of sample.
Multiplexed immunoassays advance the understanding of intracellular communication by enabling the researcher to:
- Measure multiple total or phosphorylated sites along a signaling pathway, in a single sample
- Simultaneously measure sites in multiple pathways
- Conduct all necessary measurements using minimal sample volume
However, multiplexing still presents some challenges -- there is a lack of phospho-specific antibodies that work well on the Luminex platform due to:
- Requirement for native protein conformation instead of recognition of denatured proteins on Western blots
- Complex buffer interactions with phosphoproteins
- Matrix effects as well as inter-antibody effects when multiplexed
Resulting difficulties include:
- Lack of sufficient targets for pathway analysis
- Qualitative, not quantitative results
- Needing separate wells to analyze each phosphorylated site on a specific analyte, as well as total protein.
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Successful Multiplexing with MILLIPLEX EpiQuant TechnologyTo overcome multiplexing challenges, in addition to our MILLIPLEX MAP cell signaling kits and MAPmates,Millipore proudly introduces our new MILLIPLEX EpiQuant Cell Signaling portfolio – a major advance in the design and implementation of quantitative immunoassays.
Using computational algorithms to identify unique, continuous linear sequences in proteins, EpiQuant technology generates antibodies against these specific sequences. Proteolytic digestion of your samples liberates these EpiQuant-bearing peptides (red and blue/purple above), enabling you to simultaneously measure both total protein abundance (light blue Ab) and site-specific phosphorylation (light purple) at the peptide level, using EpiQuant antibodies. These peptides are then used to generate standard curves, which results in quantitative measurements with picomolar sensitivity.
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Biological Validation for MILLIPLEX EpiQuant AnalytesAll MILLIPLEX MAP EpiQuant analytes have been validated for both biological relevance and specificity.
In the above experiments, A431 and Jurkat cells were cultured, serum-starved and stimulated with H2O2. Following lysis and digestion, aliquot samples were analyzed for total and phosphorylated Raf using the MILLIPLEX EpiQuant phosphotyrosine cell signaling panel 1. The mean fluorescence intensity (MFI) values were converted to molar concentrations using peptide standard curves.
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