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Evaluation of primary opioid receptor antibodies for use in western blotting

2012; Elsevier BV; Volume: 108; Issue: 3 Linguagem: Inglês

10.1093/bja/aes015

1471-6771

Hitomi Niwa, D.J. Rowbotham, David G. Lambert,

Neuropeptides and Animal Physiology

Editor—G-protein-coupled receptors (GPCRs) are major drug targets. As changes in GPCR mRNA may not accurately reflect changes in receptor protein, a better understanding requires quantification of protein expression. Measurement of receptor density using radioligands is one option and western blotting is another, with sensitive detection of proteins dependent on primary antibody specificity. The use of GPCR antibodies in western blotting continues to be controversial.1Michel MC Wieland T Tsujimoto G How reliable are G-protein-coupled receptor antibodies?.Naunyn Schmiedebergs Arch Pharmacol. 2009; 379: 385-388doi:10.1007/s00210-009-0395-yCrossref PubMed Scopus (238) Google Scholar, 2Bordeaux J Welsh A Agarwal S et al.Antibody validation.Biotechniques. 2010; 48: 197-209doi:10.2144/000113382Crossref PubMed Scopus (447) Google Scholar, 3Jositsch G Papadakis T Haberberger RV Wolff M Wess J Kummer W Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice.Naunyn Schmiedebergs Arch Pharmacol. 2009; 379: 389-395doi:10.1007/s00210-008-0365-9Crossref PubMed Scopus (123) Google Scholar, 4Jensen BC Swigart PM Simpson PC Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific.Naunyn Schmiedebergs Arch Pharmacol. 2009; 379: 409-412doi:10.1007/s00210-008-0368-6Crossref PubMed Scopus (135) Google Scholar, 5Huang P Liu-Chen LY Detecting the mu opioid receptor in brain following SDS-PAGE with multiple approaches.Front Biosci (Elite Ed). 2009; 1: 220-227PubMed Google Scholar, 6Couchman JR Commercial antibodies: the good, bad, and really ugly.J Histochem Cytochem. 2009; 57: 7-8doi:10.1369/jhc.2008.952820Crossref PubMed Scopus (80) Google Scholar, 7Grimsey NL Goodfellow CE Scotter EL Dowie MJ Glass M Graham ES Specific detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal!.J Neurosci Methods. 2008; 171: 78-86doi:10.1016/j.jneumeth.2008.02.014Crossref PubMed Scopus (101) Google Scholar As part of an ongoing study to determine opioid receptor expression in tissues from animals treated with opioids, we have evaluated six primary opioid receptor antibodies; two for µ-opioid peptide (MOP) and four for κ-opioid peptide (KOP) receptors in western blotting (Table 1). In the absence of tissue from knock-out animals,5Huang P Liu-Chen LY Detecting the mu opioid receptor in brain following SDS-PAGE with multiple approaches.Front Biosci (Elite Ed). 2009; 1: 220-227PubMed Google Scholar 8Saper CB An open letter to our readers on the use of antibodies.J Comp Neurol. 2005; 493: 477-478doi:10.1002/cne.20839Crossref PubMed Scopus (153) Google Scholar we have used Chinese hamster ovary (CHO) cells expressing rat MOP receptors (CHOrMOP cells) and rat KOP receptors (CHOrKOP cells) as positive controls, wild-type CHO cells lacking any opioid receptor expression (CHOWT) as the negative control, and rat brain as the test tissue sample. Where the antibody was specific for humans, we have also used CHO cells expressing human KOP receptors (CHOhKOP). Each cell line and frozen tissues were homogenized using an electro-homogenizer (UltraTurrax-T25) in ice-cold 50 mM Tris-buffer. After centrifuging homogenates, the supernatants were removed and the membranes were solubilized in ice-cold solubilization buffer (10 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate supplemented with 1 mg ml−1 soybean trypsin inhibitor, 1 mM o-phenanthroline, 1 μg ml−1 leupeptin, 1 mM iodoacetamide, and 250 μM phenylmethylsulfonyl fluoride), and then denatured by heating at 100°C for 5 min with loading buffer. At 130 V/70 min, 30–50 µg of total protein/lane was separated on 10% SDS–PAGE gel, transferred electrophoretically to a nitrocellulose membrane using a semi-dry blotter at 21 V/45 min. Non-specific binding was blocked with 5% skimmed milk in TBS-T (150 mM NaCl, 20 mM Tris–HCl, 0.05% Tween20, pH 7.5) and then the membrane was incubated with each primary opioid receptor rabbit antibody at a dilution of 1:250–5000 overnight at 4°C. After being rinsed with TBS-T, the membrane was incubated with a horseradish peroxidase-conjugated anti-rabbit IgG (whole molecule) developed in goat (Sigma-Aldrich, MO, USA) at a 1:1000 dilution, then visualized using the enhanced chemiluminescence system.Table 1Opioid receptor antibodies used. Taken from product information sheets. Species indicates the advertised reactivity (Hu, human; Ms, mouse; R, rat; Pr, Primate). Size indicates the advertised molecular weight of the protein expected. Applications are as advertised (ICC, immunocytochemistry; IHC, immunohistochemistry; WB, western blotting). The name of species marked with an asterisk indicates predicted reactionCompanyCatalogue numberNameSpeciesSize (kDa)ApplicationsAabcamAb17934Rabbit polyclobal to mu opioid receptorR, Ms*, Hu*45WBBNeuromicsRA10104Mu opioid receptor whole serum-rabbit antibodyHu, Ms, R, Pr70–80ICC, IHC,WBCMBL international corporationAT-3001Anti-κ-opioid receptor antibody, internal unconjugatedMs, R, HuNo informationWBDAcris Antibodies, Inc.AP01330PU-NPolyclonal antibody to kappa opioid receptor-aff-purifiedMs, R, Hu48WB, ELISAEabcamab10566Rabbit polyclonal to kappa opioid receptorMs, R*46WBFSigma-AldrichSAB4502046Anti-OPRK1, C-terminal antibody produced in rabbitHu, Ms, R42ELISA, WB Open table in a new tab Figure 1 provides six examples of antibodies for MOP and KOP receptors tested in our lab. Antibody A for MOP receptors did not show a band of the expected molecular weight (45 kDa) in CHOrMOP cells but did in CHOWT cells. Antibody B for MOP receptors produced numerous non-specific bands and a band of the expected molecular weight (70–80 kDa). Antibodies C–E for KOP receptors produced an expected band ∼50 kDa. However, in CHOWT cells, a similar pattern to CHOrKOP cells was observed. Antibodies C and D produced a staining pattern similar to that observed with CHOrMOP cells. Finally, antibody F for KOP receptors provided numerous non-specific bands and a band of the expected molecular weight (42 kDa) also present in negative controls. In our hands and using our protocol, we found that all six antibodies tested were not specific for the opioid receptor. When embarking on a series of new western blotting studies, we would endorse the advice of others to carefully evaluate all primary antibodies. This should ideally be done in knock-out animals, but the use of recombinant expression systems and wild-type cells may suffice. Where studies are aimed at examining a change in protein expression, a further control with protein knock-down using siRNA approaches would also seem sensible. The authors have collaborative links with University of Ferrara Peptides (UFPeptides) that is involved in the development of opioid ligands. D.G.L. holds a consultancy with Grunenthal GmbH. Our work on opioids is funded by HOPE Foundation for Cancer Research (www.hfcr.org), British Journal of Anaesthesia/Royal College of Anaesthetists.