Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Video

Updated: Oct 2, 2025

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
08:55

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Published on: December 14, 2017

15.5K

NLRP3 Phospho-residue Mapping by Phospho Dot Blots.

Sangeetha Shankar1, Zsofia A Bittner1, Alexander N R Weber2

  • 1Department of Immunology, University of Tübingen, Tübingen, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|February 25, 2022
PubMed
Summary

Mapping phosphorylation sites on proteins like NLRP3 is crucial. A phospho dot blot assay offers a quick and reliable method for screening potential phosphorylation sites, aiding in understanding protein regulation.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Nanoscale Organization of Membrane Tension during Neutrophil Extracellular Trap Formation Revealed by Fluorescence Lifetime Imaging.

Nano letters·2026
Same author

Multimodal Imaging Reveals Rapid Catecholamine Uptake and Release by Neutrophils.

Advanced science (Weinheim, Baden-Wurttemberg, Germany)·2026
Same author

Pancreatic cancer induces B cell lineage plasticity via Pax5 inhibition to sustain immunosuppression.

Cell death discovery·2026
Same author

Non-decameric NLRP3 reveals a TGN/MTOC-distal pathway of inflammasome activation.

Nature communications·2026
Same author

Neutrophils and the NLRP3 inflammasome: a tale of proteases, kinases, and inflammation.

Journal of leukocyte biology·2026
Same author

Effects of personalized vitamin D<sub>3</sub> on inflammation in colorectal cancer patients: a randomized trial.

British journal of cancer·2026

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Posttranslational modifications, such as phosphorylation, regulate protein function.
  • NLRP3 (NOD, LRR, and pyrin domain-containing protein 3) is a key inflammasome component regulated by phosphorylation.
  • Identifying phosphorylation sites on large proteins like NLRP3 is challenging.

Purpose of the Study:

  • To develop and describe an efficient screening method for mapping phosphorylation sites.
  • To validate the phospho dot blot assay for candidate phospho sites in NLRP3.

Main Methods:

  • Development of a phospho dot blot assay.
  • Utilizing in vitro kinase assays with candidate kinases (e.g., Bruton's Tyrosine Kinase - BTK).
  • Screening phosphorylated peptides using anti-phospho-tyrosine antibodies.
Keywords:
BTKDot blotNLRP3NLRP3 inflammasomePhosphorylationPosttranslational modificationTyrosine phosphorylation

More Related Videos

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate
11:31

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Published on: September 18, 2013

10.2K
Novel RNA-Binding Proteins Isolation by the RaPID Methodology
11:19

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Published on: September 30, 2016

9.1K

Related Experiment Videos

Last Updated: Oct 2, 2025

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
08:55

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Published on: December 14, 2017

15.5K
Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate
11:31

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Published on: September 18, 2013

10.2K
Novel RNA-Binding Proteins Isolation by the RaPID Methodology
11:19

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Published on: September 30, 2016

9.1K

Main Results:

  • The phospho dot blot assay provides a rapid and reliable method for screening potential phosphorylation sites.
  • The assay can be adapted to detect both tyrosine and serine/threonine phosphorylation.
  • Demonstrated utility for screening phospho sites in NLRP3.

Conclusions:

  • The phospho dot blot assay is an effective initial screening tool for mapping phosphorylation sites.
  • This method facilitates the study of posttranslational modifications in proteins like NLRP3.
  • The assay can be complemented by other techniques for comprehensive analysis.