Phospho-p62/SQSTM1 (Ser403) Cellular Kit HTRF®
This HTRF kit enables the cell-based quantitative detection of phosphorylated p62/STSQM1 as a readout of the autophagy pathway.
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No-wash
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High sensitivity
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All inclusive kit
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Low sample consumption
Overview
This HTRF cell-based assay conveniently and accurately quantifies phosphorylated p62/SQSTM1 at Ser403. p62/SQSTM1 is a ubiquitin–binding protein Involved in the regulation of autophagy. This is a cellular mechanism which enables the clearance of abnormal proteins or organelles, and it plays a role in cancer, neurodegenerative diseases, as well as cardiovascular and infectious diseases.
p62/SQSTM1 functions as a bridge between polyubiquitinated cargo protein and autophagosomes by interacting directly with both the cargo protein and a protein called LC3, which is involved in autophagosome formation. Recently, p62/SQSTM1 activation has been associated with ULK1 and mTor- induced phosphorylation on different residues, like Ser 403.
The activated autophagy pathway promotes the degradation of p62-associated ubiquitinated protein cargoes.
Benefits
- SPECIFICITY
- PRECISION
- LOW SAMPLE CONSUMPTION
Phospho-p62/SQSTM1 (Ser403) assay principle
Phospho-p62/SQSTM1 (Ser403) two-plate assay protocol
Phospho-p62/SQSTM1 (Ser403) one-plate assay protocol
Expression and phosphorylation of p62/SQSTM1 on Ser403 in untreated cells
Human HeLa and SH-SY5Y cells, and mouse Neuro2a cells were plated at 100,000 and 120,000 and 50,000 cells/well in a 96 well plate, respectively .
After an incubation of 24h at 37°C, 5% CO2, the cell culture medium was discarded, and 50µL of supplemented lysis buffer #4 (1X) added.
After 30min lysis at RT under gentle shaking, lysates were transferred into a low volume detection microplate as follows:
16µL of lysate for the detection of phospho-p62/SQSTM1 (S403)
4 µl of lysate, then 12 µL of supplemented lysis buffer #4 (1X) for the detection of total p62/SQSTM1.
Finally, 4µL of the HTRF phospho (Ser403) or total p62/SQSTM1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Whereas the expression of p62/SQSTM1 is evidenced in untreated cell lines assessed here, its phosphorylation level is elevated in Neuro2A cells only.
Beside demonstrating the compatibility of the assays with human and mouse samples, these results suggest differences in p62/SQSTM1 behavior depending on the cellular background.
Bafilomycin A1 dose-response
Human HeLa and SH-SY5Y cells were plated at respectively 100,000 and 120,000 cells/well in a 96 well plate, and incubated 24h at 37°C, 5% CO2.
After overnight incubation with increasing concentrations of Bafilomycin A1, which is a well established autophagic flux blocker, the cell culture medium was removed, and cells were lysed with 50 µL of supplemented lysis buffer #4 (1X). After 30min lysis at RT under gentle shaking, lysates were transferred into a low volume detection microplate as follows:
16µL of lysate for the detection of phospho-p62/SQSTM1 (S403).
4 µl of lysate then 12 µL of supplemented lysis buffer #4 (1X) for the detection of total p62/SQSTM1.
Finally, 4µL of the HTRF phospho (Ser403) or total p62/SQSTM1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
As expected, increasing concentrations of Bafilomycin A1 are associated with a dose dependent increase of both expression and S403 phosphorylation of p62/SQSTM1.
Note that Bafilomycin EC50 is approximately 20nM, and comparable between Hela and SH-SY5Y cells.
Starvation 6h on HeLa cells
Human HeLa cells were plated at 100,000 cells/well in a 96 well plate. After 24h incubation, cell culture medium was removed and replaced by EBSS (an amino acid and vitamin deprived medium) or fresh culture medium for 6h.
Next, cells were lysed with 50 µL of supplemented lysis buffer #4 (1X). After 30min lysis at RT under gentle shaking, lysates were transferred into a low volume detection microplate as follows:
16µL of lysate for the detection of phospho-p62/SQSTM1 (S403).
4 µl of lysate, then 12 µL of supplemented lysis buffer #4 (1X) for the detection of total p62/SQSTM1.
Finally, 4µL of the HTRF phospho (Ser403) or total p62/SQSTM1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
As expected, amino acid deprivation conditions are associated with a decrease in total p62/SQSTM1, which results from an autophagy-induced degradation mechanism.
PP242 dose-response
Human HeLa cells and SH-SY5Y cells were plated at respectively 100,000 and 120,000 cells/well in a 96 well plate.
After cell treatment (Hela: 6H; SH-SY5Y: ON) with increasing concentrations of an autophagic inducer, PP242, the culture medium was removed.
Next, cells were lysed with 50 µL of supplemented lysis buffer #4 (1X). After 30 min lysis at RT under gentle shaking, lysates were transferred into a low volume detection microplate as follows:
16 µL of lysate for the detection of phospho-p62/SQSTM1 (S403).
4 µL of lysate, then 12 µL of supplemented lysis buffer #4 (1X) for the detection of total p62/SQSTM1.
Finally, 4 µL of the HTRF phospho (Ser403) or total p62/SQSTM1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
These results show that the p62/SQSTM1 level is reduced upon PP242 treatment, resulting from a degradation induced by the autophagic pathway.
HTRF phospho-p62/SQSTM1 (Ser403) cellular assay compared to Western Blot
Human HeLa cells were grown for 2 days, until 80% confluency was reached. Then the cells were stimulated with MG-132 (4 µM) overnight, before lysis with supplemented lysis buffer, and soluble supernatants were collected via centrifugation.
Serial dilutions of the cell lysate in supplemented lysis buffer #4 (1X) were performed, and 16µL of lysates were transferred into a HTRF low volume detection microplate prior to the addition of 4µL of HTRF phospho-p62/SQSTM1 (S403) detection reagents.
A side by side comparison of Western Blot and HTRF demonstrates that the HTRF assay is 4-fold more sensitive than the Western Blot, at least under these experimental conditions.
Simplified pathway for p62/SQSTM1 assays
Autophagy is a physiological cellular process enabling the clearing out of misfolded or aggregated proteins, or damaged organelles like mitochondria, also called mitophagy. Activated upon oxidative stress, nutrient deprivation, infections (xenophagy), or during the development of cancers or neurodegenerative diseases, the autophagy pathway relies on key players such as p62/SQSTM1, ATG proteins, or LC3.
p62 / SQSTM1 is an adaptor protein which is initially phosphorylated by ULK1 on Serine 407, then on Serine 403 by casein kinase 2 or TBK1. The phosphorylation on Ser403 increases its affinity for ubiquitin chains, thus enabling p62/SQSTM1 to bind to ubiquitinated cargo proteins. Ubiquitinated proteins or ubiquitin coated mitochondria associated with p62SQSTM1 proteins are taken away in phagosomes, whose content is cleared out after lysosomal fusion.
In addition, another p62/SQSTM1 phosphorylation residue has been reported on Ser349 upon oxidative stress or amino acid deprivation. This phosphorylation event abrogates the interaction between Keap1 and the transcription factor Nrf2 which translocates into the nucleus, where it activates the transcription of antioxidant genes including p62/SQSTM1. In parallel, cytoplasmic Keap1 is captured by p62/SQSTM1 and is degraded by autophagic clearance.
Apart from the role of p62/SQSTM1 in addressing unwanted cargoes to autophagic clearance, other components are essential in the autophagy process. Among them are ULK1 and VSP34 complexes, as well as the conjugation machinery composed of the ATG protein family and LC3-II which are sequentially involved in the formation of phagophores. Phagophores evolve to autophagosomes which fuse with lysosomes, giving rise to autolysosomes where enzymes like protease or phosphatases are active.
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
HTRF® cell signaling platform combined with iCell® Hepatocytes
A solution for phospho-protein analysis in metabolic disorders - Posters
HTRF phospho-assays reveal subtle drug-induced effects
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - Posters
Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells
Analysis of a large panel of diverse biological samples and cellular models - Posters
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Videos
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Product Insert p62 phospho-S403 Kit / 64P62S4PEG-64P62S4PEH
64P62S4PEG-64P62S4PEH - Product Insert
Innovative solutions in Virology research
Kits and reagents for virology research - Flyers
Assays for neurosciences research
Advance your research on neurodegenerative diseases - Flyers
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Safety Data Sheet (DEU) p62 phospho-S403 Kit / 64P62S4PEG
64P62S4PEG - Safety Data Sheet
Safety Data Sheet (ELL) p62 phospho-S403 Kit / 64P62S4PEG
64P62S4PEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) p62 phospho-S403 Kit / 64P62S4PEG
64P62S4PEG - Safety Data Sheet
Safety Data Sheet (ITA) p62 phospho-S403 Kit / 64P62S4PEG
64P62S4PEG - Safety Data Sheet
Safety Data Sheet (SPA) p62 phospho-S403 Kit / 64P62S4PEG
64P62S4PEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) p62 phospho-S403 Kit / 64P62S4PEG
64P62S4PEG - Safety Data Sheet
Safety Data Sheet (ENG-US) p62 phospho-S403 Kit / 64P62S4PEG
64P62S4PEG - Safety Data Sheet
Safety Data Sheet (DEU) p62 phospho-S403 Kit / 64P62S4PEH
64P62S4PEH - Safety Data Sheet
Safety Data Sheet (ELL) p62 phospho-S403 Kit / 64P62S4PEH
64P62S4PEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) p62 phospho-S403 Kit / 64P62S4PEH
64P62S4PEH - Safety Data Sheet
Safety Data Sheet (ITA) p62 phospho-S403 Kit / 64P62S4PEH
64P62S4PEH - Safety Data Sheet
Safety Data Sheet (SPA) p62 phospho-S403 Kit / 64P62S4PEH
64P62S4PEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) p62 phospho-S403 Kit / 64P62S4PEH
64P62S4PEH - Safety Data Sheet
Safety Data Sheet (ENG-US) p62 phospho-S403 Kit / 64P62S4PEH
64P62S4PEH - Safety Data Sheet
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