Harvester

Manufactured by Brandel Inc

Sourced in United States

The Harvester is a specialized laboratory equipment designed for the collection and harvesting of biological samples. It functions as a precise and efficient tool for the extraction and separation of cellular components, tissues, or other biological materials from a sample. The Harvester operates through a controlled and automated process, ensuring consistent and reliable results.

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Lab products found in correlation

Radiolabeled compounds, by PerkinElmer (2 mentions) Cobra quantum gamma counter, by Hewlett-Packard (2 mentions) 125i cyp, by PerkinElmer (2 mentions) Packard cobra quantum gamma counter, by Hewlett-Packard (2 mentions) Thermomixer comfort, by Eppendorf (2 mentions) 3h olmesartan, by American Radiolabeled Chemicals (2 mentions) Gf c filters, by Brandel Inc (1 mentions) Wallac 1450 microbeta, by PerkinElmer (1 mentions) Wizard2 2 detector gamma counter, by PerkinElmer (1 mentions) Expi293f cells, by Thermo Fisher Scientific (1 mentions) Gf b glass fiber filters, by Brandel Inc (1 mentions) Ls6000 scintillation counter, by Beckman Coulter (1 mentions) Tri carb 2900tr, by PerkinElmer (1 mentions) 3h thymidine, by PerkinElmer (1 mentions) Gf b filters, by Brandel Inc (1 mentions) Gf a filter, by Cytiva (1 mentions)

19 protocols using harvester

Opioid Ligand Binding Assay Protocol

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Radiolabeled
compounds were purchased from PerkinElmer (Waltham, MA, U.S.). Opioid
ligand binding assays were performed by competitive displacement of
0.2 nM [³H]diprenorphine (250 μCi, 1.85 TBq/mmol)
by the peptidomimetic from membrane preparations containing opioid
receptors as described above. The assay mixture, containing membranes
(20 μg protein/tube) in 50 mM Tris-HCl buffer (pH 7.4), [³H]diprenorphine, and various concentrations of test peptidomimetic,
was incubated at room temperature for 1 h to allow binding to reach
equilibrium. The samples were rapidly filtered through Whatman GF/C
filters using a Brandel harvester (Brandel, Gaithersburg, MD, U.S.)
and washed five times with 50 mM Tris-HCl buffer. Bound radioactivity
on dried filters was determined by liquid scintillation counting,
after saturation with EcoLume liquid scintillation cocktail, in a
Wallac 1450 MicroBeta (PerkinElmer, Waltham, MA, U.S.). Nonspecific
binding was determined using 10 μM naloxone. The results presented
are the mean ± standard error (SE\M) from at least three separate
assays performed in duplicate. K_i (nM)
values were calculated using nonlinear regression analysis to fit
a logistic equation to the competition data using GraphPad Prism,
version 6.0c, for Mac OS X (GraphPad Software Inc., La Jolla, CA).

Harland A.A., Bender A.M., Griggs N.W., Gao C., Anand J.P., Pogozheva I.D., Traynor J.R., Jutkiewicz E.M, & Mosberg H.I. (2016). Effects of N-Substitutions on the Tetrahydroquinoline (THQ) Core of Mixed-Efficacy μ-Opioid Receptor (MOR)/δ-Opioid Receptor (DOR) Ligands. Journal of Medicinal Chemistry, 59(10), 4985-4998.

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Opioid Receptor Binding Assay Using Radiolabeled Ligand

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Radiolabeled compounds were purchased from Perkin-Elmer (Waltham, MA, U.S.). Opioid ligand binding assays were performed by competitive displacement of 0.2 nM [³H]-diprenorphine (250 μCi, 1.85 TBq/mmol) by the peptidomimetic from membrane preparations containing opioid receptors as described above. The assay mixture, containing membranes (20 μg protein/tube) in 50 mM Tris-HCl buffer (pH 7.4), [³H]-diprenorphine, and various concentrations of test peptidomimetic, was incubated at room temperature on a shaker for 1 h to allow binding to reach equilibrium. Samples were rapidly filtered through Whatman GF/C filters using a Brandel harvester (Brandel, Gaithersburg, MD, U.S.) and washed three times with 50 mM Tris-HCl buffer. Bound radioactivity on dried filters was determined by liquid scintillation counting, after saturation with EcoLume liquid scintillation cocktail, in a Wallac 1450 MicroBeta (Perkin-Elmer, Waltham, MA, U.S.). Nonspecific binding was determined using 10 μM naloxone. The results presented are the mean ± standard error (S.E.M.) from at least three separate assays performed in duplicate. Ki (nM) values were calculated using nonlinear regression analysis to fit a logistic equation to the competition data using GraphPad Prism, version 6.0c (GraphPad Software Inc., La Jolla, CA).

Nastase A.F., Anand J.P., Bender A.M., Montgomery D., Griggs N.W., Fernandez T.J., Jutkiewicz E.M., Traynor J.R, & Mosberg H.I. (2019). Dual Pharmacophores Explored via Structure-Activity Relationship (SAR) Matrix: Insights into Potent, Bifunctional Opioid Ligand Design. Journal of medicinal chemistry, 62(8), 4193-4203.

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Radioligand Binding Assay for β2AR

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Plasma membranes from the Expi293F cells (Thermo Fisher Scientific) transiently expressing each of the WT or mutant receptors were prepared as described previously (60 (link)). With each of the prepared membranes, radioligand competition binding assays were performed using a radiolabeled antagonist, [¹²⁵I]-cyanopindolol (CYP) (2,200 Ci/mmol; PerkinElmer) at a concentration of 60 pM. Reactions started upon mixing the isolated membranes together with ¹²⁵I-CYP, Cmpd-6 at varying concentrations, and a serial dilution of a competitor β₂-agonist in an assay buffer (75 mM Tris-HCl, pH 7.4, 2 mM EDTA, pH 8.0, 12.5 mM MgCl₂, 0.1% BSA, and 1 mM ascorbic acid), as indicated on each figure. Reaction mixtures were incubated for 90 minutes at ambient temperature to reach the equilibrium state. Assays were then terminated by rapid filtration of the reaction mixtures onto GF/B glass-fiber filters (Brandel) treated with 0.3% polyethyleneimine and washed with 8 mL of a cold binding buffer (75 mM Tris-HCl, pH 7.4, 2 mM EDTA, pH 8.0, 12.5 mM MgCl₂) using a harvester (Brandel). The extent of ¹²⁵I-CYP bound to the β₂AR in isolated membranes was measured using a WIZARD² 2-Detector Gamma Counter (PerkinElmer). Data were expressed as specific binding obtained by subtraction of nonspecific binding determined in the presence of high-affinity propranolol at 20 μM.

Ahn S., Maarsingh H., Walker J.K., Liu S., Hegde A., Sumajit H.C., Kahsai A.W, & Lefkowitz R.J. (2023). Allosteric modulator potentiates β2AR agonist–promoted bronchoprotection in asthma models. The Journal of Clinical Investigation, 133(18), e167337.

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Nanobody-mediated Radioligand Binding Assay

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Competition binding assays (250 µL) contained 60 pM [¹²⁵I]-CYP, a serial dilution of competitor, the indicated concentration of nanobody/Gs, and approximately 0.5 ng of β₂AR nanodiscs diluted in assay buffer (50 mM Tris-HCl pH 7.4, 12.5 mM MgCl₂, 2 mM EDTA, 0.05% BSA, 1mM L-ascorbic acid). Total binding was determined in the absence of competitor; nonspecific binding was determined using 10 µM propranolol. Following a 90 min incubation at room temperature, binding assays were terminated by rapid filtration onto GF/B glass-fiber filters treated with 0.3% PEI and washed with 8 mL of cold binding buffer using a harvester (Brandel, Gaitherburg, MD). Bound [¹²⁵I] was quantified using a Packard Cobra Quantum gamma counter (Packard, San Diego, CA) and expressed as specific binding. For [³H]-ICI 118,551 binding, 10 ng of β₂AR nanodiscs were incubated with 0.3 nM radioligand and varying concentrations of nanobody and were harvested as described above. [I¹²⁵]-CYP affinity was determined using saturation binding (Extended Data Table 1). All binding data represent a minimum of three independent experiments with deviation represented as standard error.

Staus D.P., Strachan R.T., Manglik A., Pani B., Kahsai A.W., Kim T.H., Wingler L.M., Ahn S., Chatterjee A., Masoudi A., Kruse A.C., Pardon E., Steyaert J., Weis W.I., Prosser R.S., Kobilka B.K., Costa T, & Lefkowitz R.J. (2016). Allosteric Nanobodies Reveal the Dynamic Range and Diverse Mechanisms of GPCR Activation. Nature, 535(7612), 448-452.

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Binding Affinity of V-1520 in C6 Glioma Cells

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Binding affinity of V-1520 was determined using C6 glioma cell lysates as previously reported (20 (link), 27 (link), 32 (link), 38 (link)). Lysates were incubated with N-(sec-butyl)-1-(2-chlorophenyl)-N-methyl-³H-isoquinoline-3-carboxamide ([³H]PK11195) (final concentration = 6 nM) and V-1520. The reaction was terminated by rapid filtration through a Brandel harvester and collection onto a filter presoaked with 0.3% polyethylenimine. Filters were then punched out into scintillation vials and bound radioactivity measured on a Beckman LS 6000 Scintillation Counter. All experiments were conducted in triplicate. Competition curves were analyzed with GraphPad Prism to obtain binding affinity (IC₅₀). K_i values were calculated using the equation K_i = IC₅₀/(1+[radioligand]/K_d), where IC₅₀ is determined from the competition curves, [radioligand] is the final concentration of [³H]PK11195 (6 nM) and K_d is the dissociation constant for [³H]PK11195 (5 nM).

Cohen A.S., Li J., Hight M.R., McKinley E., Fu A., Payne A., Liu Y., Zhang D., Xie Q., Bai M., Ayers G.D., Noor Tantawy M., Smith J.A., Revetta F., Kay Washington M., Shi C., Merchant N, & Manning H.C. (2020). TSPO-targeted PET and Optical Probes for the Detection and Localization of Pre-Malignant and Malignant Pancreatic Lesions. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(22), 5914-5925.

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Saturation and Competition Binding Assays for D2L Receptor

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For saturating binding assays cell membranes (c-Myc-D_2L-Flp-In CHO, 20 µg) were incubated with varying concentrations of [³H]spiperone and 10 µM haloperidol as a non-specific control, in binding buffer with (20 mM HEPES, 100 mM NaCl, 6 mM MgCl₂, 1 mM EGTA, and 1 mM EDTA, pH 7.4) or without (20 mM HEPES, 100 mM NMDG, 6 mM MgCl₂, 1 mM EGTA, and 1 mM EDTA, pH 7.4) sodium ions to a final volume of 1 mL and were incubated at 37 °C for 3 h. For competition and interaction binding assays cell membranes (c-Myc-D_2L-Flp-In CHO, 20 µg) were incubated with varying concentrations of test compound in binding buffer with (20 mM HEPES, 100 mM NaCl, 6 mM MgCl₂, 1 mM EGTA, and 1 mM EDTA, pH 7.4) or without (20 mM HEPES, 100 mM NMDG, 6 mM MgCl₂, 1 mM EGTA, and 1 mM EDTA, pH 7.4) sodium ions, containing 0.15 nM of [³H]spiperone and 100 µM GppNHp to a final volume of 1 mL and were incubated at 37 °C for 3 h. Binding was terminated by fast-flow filtration over GF/B membranes using a Brandel harvester followed by three washes with ice-cold 0.9% NaCl. Bound radioactivity was measured in a Tri-Carb 2900TR liquid scintillation counter (PerkinElmer).

Draper-Joyce C.J., Verma R.K., Michino M., Shonberg J., Kopinathan A., Klein Herenbrink C., Scammells P.J., Capuano B., Abramyan A.M., Thal D.M., Javitch J.A., Christopoulos A., Shi L, & Lane J.R. (2018). The action of a negative allosteric modulator at the dopamine D2 receptor is dependent upon sodium ions. Scientific Reports, 8, 1208.

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Nanobody-mediated Radioligand Binding Assay

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GTPγS Functional Assay for MOR

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[³⁵S]-GTPγS functional assays were conducted in the mouse MOR cell membrane used for the receptor binding assays. Membrane protein (10 μg) was incubated with varying concentrations of test compounds, GDP (15 μM), and 80 pM [³⁵S]-GTPγS in assay buffer for 1 h at 30 °C. Nonspecific binding was determined with 10 μM unlabeled GTPγS. DAMGO (5 μM) was included in the assay for a maximally effective concentration of a full agonist for the MOR. After incubation, the bound radioactive ligand was separated from the free radioligand by filtration through a GF/B glass fiber filter paper and rinsed three times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.2) using the Brandel harvester. The results were determined by utilizing a scintillation counter. All assays were determined in triplicate and repeated at least three times. Percent DAMGO stimulated [³⁵S]-GTPγS binding was defined as (net-stimulated binding by ligand/net-stimulated binding by 3 μM DAMGO) × 100%.

Cynx J., Williams H, & Nottebohm F. (1992). Hemispheric differences in avian song discrimination.. Proceedings of the National Academy of Sciences of the United States of America, 89(4), 1372-1375.

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Radioligand Binding Assay for β2AR Characterization

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Competition radioligand binding assays were performed with purified β2AR reconstituted into HDL particles (nanodiscs) and radioiodinated cyanopindolol ([¹²⁵I]-CYP (2200 Ci/mmol; PerkinElmer). Binding experiments (150 µL) contained 60 pM [¹²⁵I]-CYP, a serial dilution of competitor (isoproterenol), aptamers (2.5 µM), and β₂AR in HDL (~ 0.6 ng) diluted in assay buffer (20 mM HEPES pH 7, 20 mM NaCl, 5 mM KCl, 10 mM MgCl₂, 2 mM CaCl₂, 0.05% BSA, 1mM L-ascorbic acid). Aptamers were first heat denatured at 65 °C for 5 min and then cooled at RT. After a 90 min incubation at RT, binding assays were terminated by vacuum filtration through GF/B glass-fiber filters treated with 0.3% polyethylenimine and washed three time with cold buffer using a harvester (Brandel). Total binding was measured in the absence of competitor and nonspecific binding was determined in the presence of 10 µM propranolol. Radioligand binding was measured in a Packard Cobra Quantum gamma counter (Packard). All results are from at least three independent experiments. Fifty percent inhibitory concentrations (IC₅₀) were determined by fitting the data from the competition studies to nonlinear regression analysis (one-site competition model) using Prism (GraphPad Software).

Kahsai A.W., Wisler J.W., Lee J., Ahn S., Cahill TJ I.I.I., Dennison S.M., Staus D.P., Thomsen A.R., Anasti K.M., Pani B., Wingler L.M., Desai H., Bompiani K.M., Strachan R.T., Qin X., Alam S.M., Sullenger B.A, & Lefkowitz R.J. (2016). Conformationally Selective RNA Aptamers Allosterically Modulate the β2-Adrenoceptor. Nature chemical biology, 12(9), 709-716.

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Competitive Radioligand Binding Assay for A2BAR

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Were performed at 25 °C and in a total volume of 400 μL. CHO-spap-hA_2BAR membranes were incubated for 2 hr with the unlabeled compounds (at a final concentration of 10 × IC₅₀) while shaking at 1,000 RPM in an Eppendorf thermomixer comfort (Eppendorf AG, Hamburg, Germany). Subsequently, the samples were centrifuged at 13200 rpm (16 100g) at 4 °C for 5 min and the supernatant containing unbound ligand was removed. Pellets were resuspended in 1 mL of assay buffer, and samples were incubated for 10 min at 25 °C in the thermomixer. After four centrifugation and washing cycles in total, supernatant was discarded and the membranes were resuspended in a total volume of 400 μL containing 1.5 nM [³H]PSB-603. After 2 hr at 25 °C incubations were terminated by rapid filtration through GF/C filters using a Brandel harvester and the samples were obtained as described under “Saturation binding experiments”.

Vlachodimou A., de Vries H., Pasoli M., Goudswaard M., Kim S.A., Kim Y.C., Scortichini M., Marshall M., Linden J., Heitman L.H., Jacobson K.A, & IJzerman A.P. (2022). Kinetic profiling and functional characterization of 8-phenylxanthine derivatives as A2B adenosine receptor antagonists. Biochemical pharmacology, 200, 115027.

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