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.

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

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.

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.

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.

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.

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.