Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods
Background Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB 1 receptor in parti...
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| Published in: | Microbial cell factories Vol. 21; no. 1; pp. 1 - 18 |
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| Main Authors: | , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
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London
BioMed Central
15.09.2022
BioMed Central Ltd Springer Nature B.V BMC |
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| ISSN: | 1475-2859, 1475-2859 |
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| Abstract | Background
Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB
1
receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen.
Results
Here we generated highly soluble and stable recombinant protein constructs GST-CB1
414–472
and GST-CB1
414-442
containing much of the human CB
1
receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB
1
receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443–473 of the mouse CB
1
receptor that corresponds to residues 442–472 in the human homolog. Estimated values of CB
1
receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay.
Conclusions
Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB
1
receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints. |
|---|---|
| AbstractList | Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen.BACKGROUNDReplacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen.Here we generated highly soluble and stable recombinant protein constructs GST-CB1414-472 and GST-CB1414-442 containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay.RESULTSHere we generated highly soluble and stable recombinant protein constructs GST-CB1414-472 and GST-CB1414-442 containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay.Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints.CONCLUSIONSCollectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints. Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB.sub.1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. Here we generated highly soluble and stable recombinant protein constructs GST-CB1.sub.414-472 and GST-CB1.sub.414-442 containing much of the human CB.sub.1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB.sub.1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB.sub.1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB.sub.1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB.sub.1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints. Abstract Background Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. Results Here we generated highly soluble and stable recombinant protein constructs GST-CB1414–472 and GST-CB1414-442 containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443–473 of the mouse CB1 receptor that corresponds to residues 442–472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. Conclusions Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints. Background Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. Results Here we generated highly soluble and stable recombinant protein constructs GST-CB1414–472 and GST-CB1414-442 containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443–473 of the mouse CB1 receptor that corresponds to residues 442–472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. Conclusions Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints. Background Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB.sub.1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. Results Here we generated highly soluble and stable recombinant protein constructs GST-CB1.sub.414-472 and GST-CB1.sub.414-442 containing much of the human CB.sub.1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB.sub.1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB.sub.1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB.sub.1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. Conclusions Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB.sub.1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints. Keywords: GPCR expression analysis, Quantitative Western blot, Radioligand saturation binding, Cannabinoid CB.sub.1 receptor antibodies, Carboxy-terminal tail, Soluble recombinant protein standards, GST fusion proteins Background Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB 1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. Results Here we generated highly soluble and stable recombinant protein constructs GST-CB1 414–472 and GST-CB1 414-442 containing much of the human CB 1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB 1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443–473 of the mouse CB 1 receptor that corresponds to residues 442–472 in the human homolog. Estimated values of CB 1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. Conclusions Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB 1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints. |
| ArticleNumber | 192 |
| Audience | Academic |
| Author | Saumell-Esnaola, Miquel López de Jesús, Maider González-Burguera, Imanol Goicolea, María Aranzazu Sallés, Joan Elejaga-Jimeno, Ainhoa Echeazarra, Leyre Gómez-Caballero, Alberto Borrega-Román, Leire Barrondo, Sergio García del Caño, Gontzal |
| Author_xml | – sequence: 1 givenname: Miquel surname: Saumell-Esnaola fullname: Saumell-Esnaola, Miquel organization: Department of Pharmacology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Bioaraba, Neurofarmacología Celular y Molecular – sequence: 2 givenname: Ainhoa surname: Elejaga-Jimeno fullname: Elejaga-Jimeno, Ainhoa organization: Department of Analytical Chemistry, Faculty of Pharmacy, University of the Basque Country UPV/EHU – sequence: 3 givenname: Leyre surname: Echeazarra fullname: Echeazarra, Leyre organization: Department of Physiology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Bioaraba, Dispositivos Móviles Para El Control de Enfermedades Crónicas – sequence: 4 givenname: Leire surname: Borrega-Román fullname: Borrega-Román, Leire organization: Department of Pharmacology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Bioaraba, Neurofarmacología Celular y Molecular – sequence: 5 givenname: Sergio surname: Barrondo fullname: Barrondo, Sergio organization: Department of Pharmacology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Bioaraba, Neurofarmacología Celular y Molecular, Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM) – sequence: 6 givenname: Maider surname: López de Jesús fullname: López de Jesús, Maider organization: Department of Pharmacology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Bioaraba, Neurofarmacología Celular y Molecular – sequence: 7 givenname: Imanol surname: González-Burguera fullname: González-Burguera, Imanol organization: Bioaraba, Neurofarmacología Celular y Molecular, Department of Neurosciences, Faculty of Pharmacy, University of the Basque Country UPV/EHU – sequence: 8 givenname: Alberto surname: Gómez-Caballero fullname: Gómez-Caballero, Alberto organization: Department of Analytical Chemistry, Faculty of Pharmacy, University of the Basque Country UPV/EHU – sequence: 9 givenname: María Aranzazu surname: Goicolea fullname: Goicolea, María Aranzazu organization: Department of Analytical Chemistry, Faculty of Pharmacy, University of the Basque Country UPV/EHU – sequence: 10 givenname: Joan surname: Sallés fullname: Sallés, Joan organization: Department of Pharmacology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Bioaraba, Neurofarmacología Celular y Molecular, Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM) – sequence: 11 givenname: Gontzal surname: García del Caño fullname: García del Caño, Gontzal email: gontzal.garcia@ehu.eus organization: Bioaraba, Neurofarmacología Celular y Molecular, Department of Neurosciences, Faculty of Pharmacy, University of the Basque Country UPV/EHU |
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| Keywords | Cannabinoid CB Soluble recombinant protein standards GST fusion proteins GPCR expression analysis receptor antibodies Carboxy-terminal tail Radioligand saturation binding Quantitative Western blot |
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Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor... Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR)... Background Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor... Abstract Background Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled... |
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| SubjectTerms | Antibodies Antigens Applied Microbiology Aqueous solutions Binding Binding sites Biological properties Biological samples Biotechnology Cannabinoid CB1 receptor antibodies Cannabinoid CB1 receptors Cannabinoids Carboxy-terminal tail Cell membranes Cell receptors Chemical properties Chemistry Chemistry and Materials Science Cloning Cost analysis Density Design Enzymology G protein-coupled receptors Genetic Engineering GPCR expression analysis Homology Hydrophobicity Ligands Medical research Membrane proteins Membranes Methods Microbial Genetics and Genomics Microbiology Peptides Pharmaceutical industry Physiological aspects Plasmids Production processes Protein engineering Protein expression Proteins Quantitative analysis Quantitative Western blot Radioactivity Radioligand saturation binding Receptor density Receptors Recombinant proteins Residues Saturation Soluble recombinant protein standards Synaptosomes Transmembrane domains Western immunoblotting |
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| Title | Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods |
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