A simple, inexpensive filtration device suitable for neurotransmitter receptor binding studies in brain tissue

1981 ◽  
Vol 59 (5) ◽  
pp. 504-506 ◽  
Author(s):  
M. Wilkinson ◽  
Dale Grovestine
Keyword(s):  
Rat Brain ◽  
Receptor Binding ◽  
Radioligand Binding ◽  
Benzodiazepine Receptors ◽  
Brain Homogenate ◽  
Binding Assays ◽  
Binding Studies ◽  
Neurotransmitter Receptor ◽  
Binding Data ◽  
Filtration Device

We describe the construction and use of an inexpensive filtration manifold suitable for radioligand binding assays. Experiments with [3H]flunitrazepam indicate that the binding data for benzodiazepine receptors in rat brain homogenate are almost indistinguishable from those obtained with a commercially available manifold.

Characterisation of [3H]gabapentin binding to a novel site in rat brain: homogenate binding studies

1993 ◽  
Vol 244 (3) ◽  
pp. 293-301 ◽  
Author(s):  
Nirmala Suman-Chauhan ◽  
Louise Webdale ◽  
David R. Hill ◽  
Geoffrey N. Woodruff
Keyword(s):  
Rat Brain ◽  
Brain Homogenate ◽  
Binding Studies

scholarly journals Response of mouse proximal straight tubule and medullary thick ascending limb to beta-agonist.

1993 ◽  
Vol 4 (5) ◽  
pp. 1151-1158
Author(s):  
N S Morgunov ◽  
Y D You ◽  
D J Hirsch
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Beta Agonist ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Beta Receptor ◽  
Medullary Thick Ascending Limb ◽  
Binding Data ◽  
Straight Tubules

Scatchard analysis of (3H)CGP-12177 and (125I)cyanopindolol (CYP) radioligand binding data revealed the presence of specific beta-adrenergic receptor-binding sites on microdissected mouse proximal straight and medullary thick ascending limb (mTAL) tubules. beta-Receptor (10(-6) M isoproterenol) stimulation of isolated perfused mTAL tubules produced a consistent hyperpolarization of the transepithelial potential that was blocked by propranolol (10(-6) M), a beta-receptor antagonist, and furosemide (10(-4) M), an inhibitor of the Na+/K+/2 Cl- triporter. In contrast, there was no electrogenic response to isoproterenol stimulation in isolated perfused proximal straight tubules. In summary, radioligand binding data show that both proximal straight and mTAL tubules possess beta-receptor-binding sites. Electrogenic transport in the mTAL can be modulated by beta-agonists, but there was no detectable electrogenic response to beta-receptor stimulation in proximal straight tubules.

scholarly journals Structural Analysis, Molecular Modelling and Preliminary Competition Binding Studies of AM-DAN as a NMDA Receptor PCP-Site Fluorescent Ligand

2019 ◽  
Author(s):  
Sethu Ndzibongwana ◽  
Samukelo Ngobese ◽  
Ahmad Sayed ◽  
Ciniso Shongwe ◽  
Simon White-Phillips ◽  
...  
Keyword(s):  
Molecular Modelling ◽  
Radioligand Binding ◽  
Calcium Flux ◽  
Binding Assays ◽  
Binding Studies ◽  
Disease States ◽  
Competition Binding ◽  
Integral Role ◽  
Biological Tool ◽  
Fluorescent Ligand

Excitotoxicity related to the dysfunction of the N-methyl-d-aspartate receptor (NMDAR) has been indicated to play an integral role in the pathophysiology of multiple disease states, including neurodegenerative disorders such as Parkinson’s disease. There is a notable gap in the market for novel NMDAR antagonists, however current methods to analyze potential antagonists rely on indirect measurements of calcium flux and hazardous radioligand binding assays. Recently, a fluorescent NMDAR ligand, N-adamantan-1-yl-dimethylamino-1-naphthalenesulfonic acid, known as AM-DAN was developed by our group. Additional studies on this ligand is necessary to evaluate its potential as a biological tool in NMDAR research. Therefore, this study was aimed at conducting structural analyses, fluorescence experiments, high-accuracy NMDAR molecular modelling and NMDAR phencyclidine (PCP) site competition binding studies using AM-DAN. Results revealed that AM-DAN has appropriate structural properties, significant fluorescent ability in various solvents and is able to bind selectively and compete for the PCP-binding site of the NMDAR. Therefore, AM-DAN holds promise as a novel fluorescent ligand to measure the affinity of prospective drugs binding at the NMDAR PCP-site and may circumvent the use of radioligands.

Comparison of 3 AT1 Receptor Binding Assays: Filtration Assay, ScreenReady™ Target, and WGA Flashplate®

2005 ◽  
Vol 10 (2) ◽  
pp. 118-126 ◽  
Author(s):  
Regine M. van der Hee ◽  
Tanja Deurholt ◽  
Cindy C. Gerhardt ◽  
Els M. de Groene
Keyword(s):  
Angiotensin Ii ◽  
Receptor Binding ◽  
High Throughput Screening ◽  
Cell Membranes ◽  
Radioligand Binding ◽  
Nonspecific Binding ◽  
Binding Assays ◽  
Homogeneous Assay ◽  
Homogeneous Assays

In this article, the study of 3 different angiotensin II type 1 (AT1) receptor binding assays in terms of reproducibility, robustness, and feasibility for high-throughput screening (HTS) is described. The following methods were used: a nonhomogeneous filtration assay in a 96-well format using CHO-AT1 cell membranes and 2 homogeneous assays, which include the commercially available ScreenReady™ Target for the AT1 receptor and the wheat germ agglutinin (WGA) Flashplate®, which was coated “in-house” with the CHO-AT1 cell membranes. Receptors were labeled with [125I]-Sar1-Ile8-angiotensin II, and radioligand binding was displaced using the antagonist losartan and the natural agonist angiotensin II. Reproducible Kd, Bmax, and Ki values and good total binding/nonspecific binding (TB/NSB) ratios were obtained with both the ScreenReady™ Targets and the filtration assay, whereas the WGA Flashplates® showed unacceptably high nonspecific binding and high variation when applied as a homogeneous assay. However, when applied as a heterogeneous assay (i.e., when a wash step at the end of the assay is included), the results were significantly better. Interestingly, ligand affinities were consistently lower in Flashplate®-based assays than in the filtration assay. This may be due to the immobilization of the receptors onto the solid surface of the plate, affecting their conformation. In terms of reproducibility, robustness, and feasibility for HTS, the authors conclude that the ScreenReady™ Target plates are most suitable for AT1 receptor binding screening.

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