Sequestosome-1 ELISA Kit (P62/SQSTM1)

INTENDED USE

Human sequestosome-1 ELISA kit can measure concentrations of P62/SQSTM1 (sequestosome-1, A170, EBIAP, ORCA, OSIL, p60, P62, SQSTM1, ZIP3) present is plasma, serum, biological fluids or tissue homogenate samples. This assay has a minimum sensitivity detection limit of 0.094 ng/ml.

INTRODUCTION

Sequestosome-1 (SQSTM1), also known as p62, is a multifunctional protein that serves as a hub for various cellular signaling pathways. Originally discovered as an interacting partner of kinases, p62 is now recognized as receptor for ubiquitinated proteins destined for degradation by the autophagosome.

The ubiquitin-binding domain of p62 directly associates with ubiquitinated proteins, enabling it to shuttle these substrates for autophagic clearance. Its self-oligomerization properties via the Phox and Bem1p (PB1) domain allow p62 to cluster into larger aggregates. Hence, p62 bodies accumulate in the cell, sequestering ubiquitinated cargos until they are engulfed by the autophagosome. Additionally, it interacts with microtubule-associated protein 1 light chain 3 (LC3) on the auto-phagosomal membrane via a specific LC3-interacting region (LIR), acting as an adaptor between ubiquitinated substrates and the autophagic machinery.

Besides its role in aggrephagy, p62 serves as a signaling hub that regulates various cellular pathways integral to homeostasis and cell fate. For example, its interaction with TRAF6 results in downstream activation of NF-kB signaling and inflammation. It is also central to Nrf2-mediated transcriptional activity and the cellular stress response through its ability to displace Nrf2 from its repressor Keap1. Moreover, the intricate interplay between p62 and atypical kinases such as aPKCs underlies critical survival mechanisms like apoptosis and genome protection.

CONTENT

All reagents supplied need to be stored at 2 °C – 8 °C, unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.

• One 96-Well Plate: Pre-coated with anti-P62/SQSTM1 antibody.
• Standards: Lyophilized recombinant.
• Sample/Standard Dilution Buffer.
• Biotinylated-labelled Antibody.
• Antibody Dilution Buffer.
• HRP-Streptavidin Conjugate (SABC).
• SABC Dilution Buffer.
• TMB Substrate.
• Wash Buffer (25x).
• Plate Sealer.
• Product Instructions.

TYPICAL RESULTS

For this sequestosome-1 ELISA kit, it is recommended that a standard curve is generated for each assay carried out.

Standard Curve: 0, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10 ng/ml.
Reactivity: Human
Sensitivity: 0.094 ng/ml
Range: 0.156 – 10 ng/ml
Principle: Sandwich, Double Antibody
Application: Research Use Only.

ASSAY CHARACTERISTICS

– Specificity: Highly specific for P62/SQSTM1, no cross reactivity or interference between P62/SQSTM1 and analogues was detected.
– Recovery: Serum (87 – 97%), EDTA Plasma (87 – 104%), Heparin Plasma (98 – 104%).
– Linearity: Serum (88 – 98%), EDTA Plasma (89 – 101%), Heparin Plasma (86 – 102%).
– Precison Intra-Assay: CV < 8%
– Precison Inter-Assay: CV < 10%

REFERENCES

1. Regulation of selective autophagy: the p62/SQSTM1 paradigm. Essays Biochem. (2017) 61 (6): 609-624. Lamark T., et al.
2. Requirement for p62 acetylation in the aggregation of ubiquitylated proteins under nutrient stress. Nat Commun. (2019) 10 (1): 5792. You Z., et al.
3. SPOP mutations promote p62/SQSTM1-dependent autophagy and Nrf2 activation in prostate cancer. Cell Death Differ. (2022) 29 (6): 1228-1239. Shi Q., et al.
4. p62 as a therapeutic target for tumor. Eur J Med Chem. (2020) 193: 112231. Tao M., et al.

ADDITIONAL INFORMATION

• Full Name: Sequestosome-1 ELISA Kit (P62/SQSTM1)
• Reactivity: Human
• Sample Type: Plasma, Tissue Homogenates, Serum, Biological Fluids
• Sensitivity: 0.094 ng/ml

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