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HTRF Human and Mouse Total p62/SQSTM1 Detection Kit, 500 Assay Points

P62/sqstm1 Total Kit - 50,000 Tests

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Part Number: 64N62PEG
Unit Size: 500 Assay Points
Part Number: 64N62PEH
Unit Size: 10,000 Assay Points
  • This HTRF cell-based assay conveniently and accurately monitors the expression level of p62/SQSTM1 proteins, both phosphorylated and unphosphorylated. It uses the same buffers as our Phospho-p62/SQSTM1 kit, and enables the analysis of phosphorylated and total proteins from a single sample as a pivotal readout of the autophagy pathway. Autophagy is a cellular mechanism which enables the clearance of abnormal proteins or organelles, and plays a role in cancer, neurodegenerative diseases, as well as cardiovascular and infectious diseases.

    p62/SQSTM1 is a ubiquitin-binding protein and functions as a bridge between polyubiquitinated cargo protein and autophagosomes. p62 interacts directly with both the cargo protein and a protein called LC3 which is involved in the autophagosome formation. Finally, the activated autophagy pathway promotes the degradation of p62-associated ubiquitinated protein cargoes.

  • Assay Points
    500
    Assay Target Type
    Kit
    Assay Technology
    HTRF
    Brand
    HTRF
    Quantity
    1
    Therapeutic Area
    Cardiovascular
    Infectious Diseases
    Neuroscience
    Oncology & Inflammation
    Unit Size
    500 Assay Points
  • Total p62/SQSTM1 assay principle

    The Total-p62/SQSTM1 assay quantifies the expression level of p62 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The Total-p62/SQSTM1 assay uses two labeled antibodies, one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of p62/SQSTM1 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

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    Total-p62/SQSTM1 two-plate assay protocol

    The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Total-p62/SQSTM1 detection reagents. This protocol enables the cells' viability and confluence to be monitored

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    Total-p62/SQSTM1 (Ser403) one-plate assay protocol

    Detection of Total p62/SQSTM1 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

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  • Expression and phosphorylation of p62/SQSTM1 on Ser403 in untreated cells

    Human HeLa and SH-SY5Y cells, and mouse Neuro2a cells were plated at respectively 100,000 and 120,000 and 50,000 cells/well in a 96 well plate.

    After an incubation of 24h at 37°C, 5% CO2, the cell culture medium was discarded, and 50µL of supplemented lysis buffer #4 (1X) was 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 the untreated cell lines assessed here, its phosphorylation level is elevated in Neuro2A cells only.

    Beside demonstrating the compatibility of both assays with human and mouse samples, these results suggest differences in p62/SQSTM1 behavior depending on the cellular background.

    assay-validation-p62-total-1

     

    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 o, both expression and S403 phosphorylation of p62/SQSTM1.

    Note that Bafilomycin EC50 is approximately 20nM, and comparable between Hela and SH-SY5Y cells.

    assay-validation-p62-total-2

     

    assay-validation-p62-total-3

     

    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.

    assay-validation-p62-total-4

     

    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.

    assay-validation-p62-total-5

     

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    HTRF Total p62/SQSTM1 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 an HTRF low volume detection microplate prior to the addition of 4µL of HTRF total p62/SQSTM1 detection reagents.

    A side by side comparison of Western Blot and HTRF demonstrates the HTRF assay is 4-fold more sensitive than the Western Blot, at least under these experimental conditions.

    A side by side comparison of Western Blot and HTRF demonstrated that the HTRF assay is 8-fold more sensitive than the Western Blot.

    assay-validation-p62-total-7

     

  • Simplified pathway for p62/SQSTM1 assays

    Autophagy is a physiological cellular process enabling the clearing out of misfolded or aggregated proteins, and 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 the 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.

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This guide provides you an overview of HTRF applications in several therapeutic areas.