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7TM Phosphorylation Assay Kits

Analysis of agonist-driven GPCR phosphorylation provides insights into the receptor activation state and ligand pharmacology. The 7TM Phosphorylation Assay is the first-in-class immunoassay for the quantitative assessment of GPCR phosphorylation that can be performed entirely in multiwell cell culture plates thus eliminating the need for Western blot analysis. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by detection using Premium Phosphosite-Specific 7TM Antibodies as phospho-biosensors (Figure 1). The 7TM Receptor Phosphorylation Assay allows quantitative determination of GPCR phosphorylation particular when total receptor is determined in parallel using Non-Phospho 7TM Antibodies, which are included in each kit. The 7TM Receptor Phosphorylation Assay can be performed using transiently or stably transfected cells. This assay protocol has been optimized for transiently transfected HEK293 cells in 96-well format using plasmids expressing 3xHA-tagged GPCR constructs. It can be adapted to other affinity tags such as FLAG, Myc, His or GFP. The assay can be performed manually or fully automated in medium- to high-throughput mode. The 7TM Phosphorylation Assay is fast, robust and reliable, thus fulfilling all the requirements for extensive application in academic and pharmaceutical research.



Figure 1. Step-by-step flowchart showing the 7TM phosphorylation assay protocol. (1) Cells expressing affinity-tagged GPCRs are grown in F-bottom cell culture plates, and upon reaching ≥ 95% confluency, the cells are exposed to the agonist, antagonist or inhibitor of interest. (2) The cells are lysed in detergent buffer, and lysates are cleared by centrifugation. (3) For parallel detection of phosphorylated and total receptors, the lysate of each sample is divided and transferred into two corresponding wells of U-bottom assay plates. (4) Anti-tag magnetic beads are added to each well for receptor immunoprecipitation. (5) Primary phosphosite-specific and phosphorylation-independent antibodies are added to the appropriate wells of each sample. (6) A secondary enzyme-labeled antibody is then added. (7) An enzyme substrate solution is added for detection, the color reaction is stopped by adding a stop solution. (8) Determine the optical density at 405 nm with a microplate reader. (9) Calculate concentration-response-curves. 


 


Figure 2. Schematic model of the recommended 7TM phosphorylation assay setup to determine concentration-response-curves of agonists or antagonists. Cells are grown in 96-F-bottom well culture plates (left panel). Cells are lysed and each lysate is divided and transferred into two corresponding wells of U-bottom assay plates (right panel). Thus, each well used for detection of phosphorylated receptor (phospho-site) has a corresponding well used for detection of total receptor as a loading control (non-phospho) (e.g. A1 and C1, etc.). Rows A to D show a typical example for agonist stimulation (green). Rows E to H show a typical example for antagonist challenge (red). Standard agonist stimulation is depicted in gray. Rows A and B as well as E and F are detected using phosphosite-specific antibodies. Rows C and D as well as G and H are used for detection of total receptors using phosphorylation-independent antibodies. Columns 1 and 2 are used for stimulation with standard agonist. Columns 3 to 9 are used for a 7-point concentration-response curve. If required columns 10 and 11 are available control stimulations. Column 12 serves as negative control in which no primary antibody is added resulting in background signal. Each stimulation is performed in duplicate.

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OD signals in pT360/pS364-β2 Phosphorylation Assay
pT360/pS364-β2 Phosphorylation Assay Kit
The pS355/pS356-β2 Phosphorylation Assay Kit allows determination of ß2 Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
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pT342-C5a1 Phosphorylation Assay Kit
pT342-C5a1 Phosphorylation Assay Kit
The pS375-MOP Phosphorylation Assay Kit allows determination of MOP Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
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pS338/pT339-C5a1 Phosphorylation Assay Kit
pS338/pT339-C5a1 Phosphorylation Assay Kit
The pS375-MOP Phosphorylation Assay Kit allows determination of MOP Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
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pS332/pS334-C5a1 Phosphorylation Assay Kit
pS332/pS334-C5a1 Phosphorylation Assay Kit
The pS375-MOP Phosphorylation Assay Kit allows determination of MOP Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
50
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pT324/pS327-C5a1 Phosphorylation Assay Kit
pT324/pS327-C5a1 Phosphorylation Assay Kit
The pS375-MOP Phosphorylation Assay Kit allows determination of MOP Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
50
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pS338/pT339-NK1 Phosphorylation Assay Kit
pS338/pT339-NK1 Phosphorylation Assay Kit
The pT370-MOP Phosphorylation Assay Kit allows determination of MOP Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
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pT356/pT357-NK1 Phosphorylation Assay Kit
pT356/pT357-NK1 Phosphorylation Assay Kit
The pT370-MOP Phosphorylation Assay Kit allows determination of MOP Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
50
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pT344/pS347-NK1 Phosphorylation Assay Kit
pT344/pS347-NK1 Phosphorylation Assay Kit
The pT370-MOP Phosphorylation Assay Kit allows determination of MOP Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
50
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OD signals in pS335/pS336-CB2 Phosphorylation Assay
pS335/pS336-CB2 Phosphorylation Assay Kit
The pS335/pS336-CB2 Phosphorylation Assay Kit allows determination of CB2 Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
50
NEW
OD signals in pT370-MOP Phosphorylation Assay
pT370-MOP Phosphorylation Assay Kit
The pT370-MOP Phosphorylation Assay Kit allows determination of MOP Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
50
NEW
OD signals in pS375-MOP Phosphorylation Assay
pS375-MOP Phosphorylation Assay Kit
The pS375-MOP Phosphorylation Assay Kit allows determination of MOP Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
50
NEW
OD signals in pT376-MOP Phosphorylation Assay
pT376-MOP Phosphorylation Assay Kit
The pT376-MOP Phosphorylation Assay Kit allows determination of MOP Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
50
NEW
OD signals in pT379-MOP Phosphorylation Assay
pT379-MOP Phosphorylation Assay Kit
The pT379-MOP Phosphorylation Assay Kit allows determination of MOP Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
50
NEW
OD signals in pS355/pS356-β2 Phosphorylation Assay
pS355/pS356-β2 Phosphorylation Assay Kit
The pS355/pS356-β2 Phosphorylation Assay Kit allows determination of ß2 Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
50
NEW
OD signals in pT338/pT340-CB2 Phosphorylation Assay
pT338/pT340-CB2 Phosphorylation Assay Kit
The pT338/pT340-CB2 Phosphorylation Assay Kit allows determination of CB2 Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
50
NEW
OD signals in pS341/pS343-SST2 Phosphorylation Assay
pS341/pS343-SST2 Phosphorylation Assay Kit
The pS341/pS343-SST2 Phosphorylation Assay Kit allows determination of SST2 Phosphorylation in 96-well plates without the need for Western blot analysis.
$ 300.00 $ 600.00 *
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For further reading refer to:

Johanna Kaufmann, Nina Kathleen Blum, Falko Nagel, Anna Schuler, Julia Drube, Carsten Degenhart, Julian Engel, Jan Eicke Eickhoff, Pooja Dasgupta, Sebastian Fritzwanker, Maria Guastadisegni, Clemens Schulte, Elke Miess-Tanneberg, Hans Michael Maric, Mariana Spetea, Andrea Kliewer, Matthias Baumann, Bert Klebl, Rainer K. Reinscheid, Carsten Hoffmann, Stefan Schulz. A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening. bioRxiv 2022.07.25.501346; doi: https://doi.org/10.1101/2022.07.25.501346

Kliewer A, Reinscheid RK, Schulz S. Emerging Paradigms of G Protein-Coupled Receptor Dephosphorylation. Trends Pharmacol Sci. 2017 Jul;38(7):621-636. doi:10.1016/j.tips.2017.04.002. Epub 2017 May 4. Review. PubMed PMID: 28478994.

Miess E, Gondin AB, Yousuf A, Steinborn R, Mösslein N, Yang Y, Göldner M, Ruland JG, Bünemann M, Krasel C, Christie MJ, Halls ML, Schulz S, Canals M. Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid μ-opioid receptor desensitization. Sci Signal. 2018 Jul 17;11(539). pii: eaas9609. doi: 10.1126/scisignal.aas9609. PubMed PMID: 30018083.

Kliewer A, Schmiedel F, Sianati S, Bailey A, Bateman JT, Levitt ES, Williams JT, Christie MJ, Schulz S. Phosphorylation-deficient G-protein-biased μ-opioid receptors improve analgesia and diminish tolerance but worsen opioid side effects. Nat Commun. 2019 Jan 21;10(1):367. doi: 10.1038/s41467-018-08162-1. PubMed PMID: 30664663; PubMed Central PMCID: PMC6341117.

Mann A, Moulédous L, Froment C, O'Neill PR, Dasgupta P, Günther T, Brunori G, Kieffer BL, Toll L, Bruchas MR, Zaveri NT, Schulz S. Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists. Sci Signal. 2019 Mar 26;12(574). pii: eaau8072. doi: 10.1126/scisignal.aau8072. PubMed PMID: 30914485; PubMed Central PMCID: PMC6934085.

Saaber F, Schütz D, Miess E, Abe P, Desikan S, Ashok Kumar P, Balk S, Huang K, Beaulieu JM, Schulz S, Stumm R. ACKR3 Regulation of Neuronal Migration Requires ACKR3 Phosphorylation, but Not β-Arrestin. Cell Rep. 2019 Feb 5;26(6):1473-1488.e9. doi: 10.1016/j.celrep.2019.01.049. PubMed PMID: 30726732.

Glück L, Loktev A, Moulédous L, Mollereau C, Law PY, Schulz S. Loss of morphine reward and dependence in mice lacking G protein-coupled receptor kinase 5. Biol Psychiatry. 2014 Nov 15;76(10):767-74. doi: 10.1016/j.biopsych.2014.01.021. Epub 2014 Feb 3. PubMed PMID: 24629717; PubMed Central PMCID: PMC4119866.

For further reading refer to: Johanna Kaufmann, Nina... read more »
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7TM Phosphorylation Assay Kits

For further reading refer to:

Johanna Kaufmann, Nina Kathleen Blum, Falko Nagel, Anna Schuler, Julia Drube, Carsten Degenhart, Julian Engel, Jan Eicke Eickhoff, Pooja Dasgupta, Sebastian Fritzwanker, Maria Guastadisegni, Clemens Schulte, Elke Miess-Tanneberg, Hans Michael Maric, Mariana Spetea, Andrea Kliewer, Matthias Baumann, Bert Klebl, Rainer K. Reinscheid, Carsten Hoffmann, Stefan Schulz. A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening. bioRxiv 2022.07.25.501346; doi: https://doi.org/10.1101/2022.07.25.501346

Kliewer A, Reinscheid RK, Schulz S. Emerging Paradigms of G Protein-Coupled Receptor Dephosphorylation. Trends Pharmacol Sci. 2017 Jul;38(7):621-636. doi:10.1016/j.tips.2017.04.002. Epub 2017 May 4. Review. PubMed PMID: 28478994.

Miess E, Gondin AB, Yousuf A, Steinborn R, Mösslein N, Yang Y, Göldner M, Ruland JG, Bünemann M, Krasel C, Christie MJ, Halls ML, Schulz S, Canals M. Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid μ-opioid receptor desensitization. Sci Signal. 2018 Jul 17;11(539). pii: eaas9609. doi: 10.1126/scisignal.aas9609. PubMed PMID: 30018083.

Kliewer A, Schmiedel F, Sianati S, Bailey A, Bateman JT, Levitt ES, Williams JT, Christie MJ, Schulz S. Phosphorylation-deficient G-protein-biased μ-opioid receptors improve analgesia and diminish tolerance but worsen opioid side effects. Nat Commun. 2019 Jan 21;10(1):367. doi: 10.1038/s41467-018-08162-1. PubMed PMID: 30664663; PubMed Central PMCID: PMC6341117.

Mann A, Moulédous L, Froment C, O'Neill PR, Dasgupta P, Günther T, Brunori G, Kieffer BL, Toll L, Bruchas MR, Zaveri NT, Schulz S. Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists. Sci Signal. 2019 Mar 26;12(574). pii: eaau8072. doi: 10.1126/scisignal.aau8072. PubMed PMID: 30914485; PubMed Central PMCID: PMC6934085.

Saaber F, Schütz D, Miess E, Abe P, Desikan S, Ashok Kumar P, Balk S, Huang K, Beaulieu JM, Schulz S, Stumm R. ACKR3 Regulation of Neuronal Migration Requires ACKR3 Phosphorylation, but Not β-Arrestin. Cell Rep. 2019 Feb 5;26(6):1473-1488.e9. doi: 10.1016/j.celrep.2019.01.049. PubMed PMID: 30726732.

Glück L, Loktev A, Moulédous L, Mollereau C, Law PY, Schulz S. Loss of morphine reward and dependence in mice lacking G protein-coupled receptor kinase 5. Biol Psychiatry. 2014 Nov 15;76(10):767-74. doi: 10.1016/j.biopsych.2014.01.021. Epub 2014 Feb 3. PubMed PMID: 24629717; PubMed Central PMCID: PMC4119866.

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