Mechanisms of Immune Regulation: Capturing Signaling Patterns through Global Phosphorylation Profiling and Intracellular Flow Cytometry
Matthew Stokes, PhD & Christopher Manning
Tweet
Transcript
Embed
Email
Bookmark
<<
<
>
>>
Tweet
Facebook
Email
Chapters
Mechanisms of Immune Regulation in Cancer -Matthew Stokes
About Cell Signaling Technology
PTMScan
®
Services
PTMScan
®
Kits
Motif / PTM Antibodies Empower PTMScan
®
The PTMScan
®
Method
PTMScan
®
Phosphorylation Enrichment Methods
PTMScan
®
in Action: TCR Signaling
Qualitative Analysis of TCR Signaling
Critical Signaling Protein Coverage by Enrichment
Quantitative Analysis of TCR Signaling
Validation of PTMScan® Data
Taking the Next Step: PTMScan Targeted Assays
Proteomics Conclusions
Mastering Phospho-Flow: Optimizing Immune Cell Activation Assays with Intra- & Extracellular Flow Cytometry-Christopher Manning
Topics
Why would I want to perform phospho-flow?
Phosphorylation is commonly detected by western blot, which has limitations
Live-cell flow cytometry is not compatible with immuno-labeling of intracellular proteins, including phosphorylation sites
What protocol should I use for phospho-flow?
A variety of fixation/permeabilization protocols will work for detection of phospho-proteins; start with a recommended protocol
Permeabilization reagents: a major difference among protocols
Your protocol options are dictated by the antibody and the target
Protocol flexibility exists for many antibodies, across multiple target types
Why isn’t this antibody working in my protocol?
Protocol changes may impact antibody specificity
Timing is critical for the detection of phospho-proteins
Timing is critical for the detection of phospho-proteins
Order of fixation and staining steps can be altered to improve efficiency and performance
CD marker labeling may be impacted by reagents
Protocol compatibility-Human-reactive CD markers, analyzed on PBMCs
Protocol compatibility-Mouse-reactive CD markers, analyzed on splenocytes or bone marrow
Some fluorophores are impacted by organic solvents
You can analyze cell signaling by Flow Cytometry!
No transcript for this webinar
More Information
Request info