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Gf b filters

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GF/B filters are a type of laboratory equipment used for filtration. They are composed of glass microfibers and designed to efficiently separate particles and retain solid materials from liquids or gases.

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Lab products found in correlation

Protease inhibitor mixture, by Roche (2 mentions) Brandel cell harvester, by Brandel Inc (2 mentions) Prism 4, by GraphPad (2 mentions) 3h ryanodine, by PerkinElmer (1 mentions) M24 r harvester, by Brandel Inc (1 mentions) Unlabeled ryanodine, by MP Biomedicals (1 mentions) 48 well harvester, by Brandel Inc (1 mentions) Microbeta jet, by PerkinElmer (1 mentions) Prism 9, by GraphPad (1 mentions) M24r cell harvester, by Brandel Inc (1 mentions) 3h ebob, by PerkinElmer (1 mentions) Ls6500 liquid scintillation counter, by Beckman Coulter (1 mentions) Gtpγs, by Thermo Fisher Scientific (1 mentions) Gtpγs, by Brandel Inc (1 mentions) 35s gtpγs, by PerkinElmer (1 mentions) M 24 cell harvester, by Brandel Inc (1 mentions) Cobra 2 γ counter, by PerkinElmer (1 mentions) Prism 7, by GraphPad (1 mentions) Tri carb 2810tr, by PerkinElmer (1 mentions) Microbeta2 lumijet, by PerkinElmer (1 mentions)

19 protocols using gf b filters

1

Radioligand Binding Assay for Ryanodine Receptors

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Binding assays were carried out following a protocol previously described (Federico et al., 2017 (link)). Binding mixtures contained 100 µg of protein from homogenates prepared from pooled atria (5–7 mice), 0.2 M KCl, 20 mM Hepes (pH 7.4), 6.5 nM [3H]ryanodine (PerkinElmer), 1 mM EGTA and enough CaCl2 to set free [Ca2+] between 10 nM (pCa2+ 8) and 100 µM (pCa2+ 4). The ratio between Ca2+ and EGTA was determined using MaxChelator (WEBMAXCLITE v1.15 http://maxchelator.stanford.edu/webmaxc/webmaxclite115.htm). Following a 2 hr incubation at 36°C, reactions were filtered through Whatman GF/B Filters using a Brandel M24-R Harvester. [3H]ryanodine binding was determined using a Beckman LS6500 scintillation counter and BioSafe II scintillation cocktail (RPI Corp). Non-specific binding was quantified in the presence of 2 µM unlabeled ryanodine (MP Biomedicals) and subtracted.
Dai W., Laforest B., Tyan L., Shen K.M., Nadadur R.D., Alvarado F.J., Mazurek S.R., Lazarevic S., Gadek M., Wang Y., Li Y., Valdivia H.H., Shen L., Broman M.T., Moskowitz I.P, & Weber C.R. (2019). A calcium transport mechanism for atrial fibrillation in Tbx5-mutant mice. eLife, 8, e41814.
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2

Radioligand Binding Assay for Opioid Receptors

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Membrane protein (40 μg) was incubated in 0.5ml of 50 mM Tris, 0.5% BSA and ~0.8nM [3H]-DPN (for CHOhMu, CHOhDelta and CHOhKappa) or ~0.8nM [3H]-UFP-101 (for CHONOP cells), as well as varying concentrations (10 μM-1pM) of the reference ligand DeNo. Non-specific binding was determined in the presence of 10 μM naloxone for CHOhMu, CHOhDelta and CHOhKappa or 1μM of N/OFQ for CHONOP cells. Samples were incubated for 1 hr at room temperature and reactions were terminated by vacuum filtration, onto PEI-soaked Whatman GF/B filters, using a Brandel harvester. The concentration of displacing ligand producing 50% displacement was corrected for the competing mass of radioligand to yield pKi, a measure of its affinity [3 (link)].
Bird M.F., Cerlesi M.C., Brown M., Malfacini D., Vezzi V., Molinari P., Micheli L., Mannelli L.D., Ghelardini C., Guerrini R., Calò G, & Lambert D.G. (2016). Characterisation of the Novel Mixed Mu-NOP Peptide Ligand Dermorphin-N/OFQ (DeNo). PLoS ONE, 11(6), e0156897.
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3

Receptor Reconstitution and Competition Binding Assay

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Purified M2R receptor was reconstituted into high-density lipoprotein (HDL) particles constituting apolipoprotein A1(APOA1) and mixture of POPG: POPC lipids with 3:2 (mol:mol)ratio as previously described74 (link). For competition binding studies, Sf9 membranes or M2R receptor reconstituted in HDL particle containing 50-100 femtomoles were incubated with 2 nM [3H]-NMS, in the presence or absence of heterotrimeric GoA, and increasing concentration of test ligand (ACh or Ixo) in a buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl and 0.5% bovine serum albumin. GoA titration competition binding assay was carried out by incubating the Sf9 membranes with a fixed concentration of [3H]-NMS and agonist with varying concentration of purified GoA for 2 h. Membranes or HDL particles were separated from excess [3H]-NMS by Whatman GF/B filters using a Brandel 48-well harvester. The bound radioligand were read on a liquid scintillation counter (MicroBeta Jet, PerkinElmer). Data were analyzed by GraphPad Prism 9.2.0.
Xu J., Wang Q., Hübner H., Hu Y., Niu X., Wang H., Maeda S., Inoue A., Tao Y., Gmeiner P., Du Y., Jin C, & Kobilka B.K. (2023). Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature Communications, 14, 376.
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4

Dissociation Kinetics of 18F-5-OH-FPPAT

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Dissociation kinetics was carried out under similar concentrations of tissue and 18F-5-OH-FPPAT (37 kBq/cc). The mixtures were incubated for 1 hr at 25°C. The dissociation of 18F-5-OH-FPPAT was initiated by adding an excess of dopamine (10 μM final concentration), 5-OH-FPPAT (10 μM final concentration), or Gpp(NH)p (50 μM final concentration) in time range of 1–60 min. The incubation was terminated by filtration through Whatman GF/B filters presoaked in 0.3% polyethylenimine, with a Brandel model M-24R cell harvester. The filters were rinsed for 10 s with ice-cold Tris-HCl buffer, and the filters were counted for fluorine-18 using an Auto-Gamma 5000 (Packard Instruments).
Mukherjee J., Majji D., Kaur J., Constantinescu C.C., Narayanan T.K., Shi B., Nour M.T, & Pan M.L. (2016). PET radiotracer development for imaging high-affinity state of dopamine D2 and D3 receptors: Binding studies of fluorine-18 labeled aminotetralins in rodents. Synapse (New York, N.Y.), 71(3), 10.1002/syn.21950.
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5

Opioid Receptor Binding Assay

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LDM, 0.025% Brij-58-treated LDM or Percoll-purified PM were incubated in TME buffer with increasing concentrations of [3H]DADLE (0.2–39.3 nM) for 60 min at 30°C. Non-specific binding was determined in the presence of 10 μM DADLE. Separation of bound and free radioactivity was performed by filtration through Whatman GF/B filters in Brandel cell harvester, the filters were washed three times with 3 ml of ice-cold incubation buffer and radioactivity was determined by liquid scintillation. Binding data were fitted with the rectangular hyperbola and Kd and Bmax values determined by GraphPad Prism4.
Roubalova L., Vosahlikova M., Brejchova J., Sykora J., Rudajev V, & Svoboda P. (2015). High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments. PLoS ONE, 10(8), e0135664.
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6

EBOB Displacement Binding Assay

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Membrane suspensions were thawed and centrifuged in incubation buffer at 10,000×g for 10 min and washed by a similar centrifugation. For displacement studies, membrane suspensions (5 mg original wet wt./ml) were incubated with 0.5 nM [3H]EBOB (26.2 µCi/mmol, Perkin Elmer Life Sciences, Boston, MA, USA) in the absence or presence of 50 µM of new compounds or reference drug for 2 h at 25°C. Nonspecific binding was determined in the presence of 10 µM Bicuculline. Bicuculline and picrotoxin were frequently used to determine the nonspecific binding when [3H]EBOB as radioligand [17] . Triplicate 1 ml samples were filtered on Whatman GF/B filters under vacuum with a Brandel Harvester and washed with 3×3 ml ice-cold buffer. Radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter.
Zhang H., Xu X., Chen Y., Qiu Y., Liu X., Liu B.F, & Zhang G. (2014). Synthesis and Evaluation of Fluorine-Substituted Phenyl Acetate Derivatives as Ultra-Short Recovery Sedative/Hypnotic Agents. PLoS ONE, 9(5), e96518.
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7

CB1 Receptor Binding Assay with [3H] CP-55,940

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The CB1R-binding assay was performed with [3H] CP-55,940 as the labeled ligand. A rapid filtration–binding assay was followed by a procedure as described previously (Basavarajappa et al., 1998 (link), 2006 (link)). HP and NC from P7 saline- and ethanol-treated mice were isolated and homogenized in an ice-cold homogenization buffer (Basavarajappa et al., 2014 (link); Basavarajappa and Subbanna, 2014 (link)) containing a freshly added 1% protease inhibitor mixture (Roche). Plasma membranes (PMs) were prepared as described previously (Basavarajappa et al., 1998 (link), 2006 (link)) and appropriate ali-quots were stored at -80°C until use. Assay solutions were incubated in silicone-treated tubes for 60 min at 30°C, with a final assay volume of 0.5 ml containing 4 nM [3H] CP-55,940 and a final PM concentration of 0.02 mg protein/ml. Nonspecific binding was determined in the presence of 30 μM CP-55,940 and was subtracted to yield specific binding values. Bound [3H] CP-55,940 was harvested by vacuum filtration through Whatman GF/B filters with a Brandel Cell Harvester. Radioactivity was determined by liquid scintillation counting after overnight incubation of filters in 5 ml of scintillation mixture UniverSol.
Subbanna S., Nagre N.N., Umapathy N.S., Pace B.S, & Basavarajappa B.S. (2015). Ethanol Exposure Induces Neonatal Neurodegeneration by Enhancing CB1R Exon1 Histone H4K8 Acetylation and Up-regulating CB1R Function causing Neurobehavioral Abnormalities in Adult Mice. International Journal of Neuropsychopharmacology, 18(5), pyu028.
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8

CB1R Membrane Ligand Binding Assay

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Membranes were prepared from 3T3-L1 cells endogenously expressing CB1R, and displacement ligand binding assays were carried out as previously described8 (link) with minor modifications. Briefly, membranes (250 μg) were incubated with [3H] SR141716A (3 nM) in 50 mM Tris-Cl, pH 7.8, containing 1 mM EGTA, 5 mM MgCl2, and protease inhibitor cocktail in the presence of Pep19 (0–10 μM) for 1 h at 30 °C. At the end of the incubation period, membranes were filtered using a Brandel filtration system and GF/B filters (presoaked for 2 h at room temperature with 0.1% polyethyleneimine containing 0.2% fatty acid free BSA), filters were washed 3 times with ice-cold 50 mM Tris-Cl (pH 7.4) and bound radioactivity measured using a liquid scintillation counter.
Reckziegel P., Festuccia W.T., Britto L.R., Jang K.L., Romão C.M., Heimann J.C., Fogaça M.V., Rodrigues N.S., Silva N.R., Guimarães F.S., Eichler R.A., Gupta A., Gomes I., Devi L.A., Heimann A.S, & Ferro E.S. (2017). A novel peptide that improves metabolic parameters without adverse central nervous system effects. Scientific Reports, 7, 14781.
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9

GTPγS Binding Assay for G-Protein Activation

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[35S]GTPγS (Perkin-Elmer), GDP (Sigma-Aldrich), and GTPγS (Fisher Scientific) were dissolved in GTPγS binding buffer (50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 0.1% BSA, 1 mM DTT, pH 7.4). 10–20 μg membrane proteins from in vitro JNK kinase assay treated preparations were incubated with 1 μM DAMGO, 1 μM U69,593, 1 μM GTPγS, or an equivalent volume of GTPγS binding buffer in 50 mM GTPγS binding buffer) at 30 °C for 1 h in the presence of 0.1 nM [35S]GTPγS and 10 mM GDP. Bound [35S]GTPγS was separated from free [35S]GTPγS by rapid filtration using a Brandel cell harvester onto GF/B filters (Brandel) pre-soaked in 50 mM Tris/0.1%BSA. Filters were washed 3x using 50 mM Tris/0.1%BSA. Bound [35S]GTPγS was measured using a liquid scintillation counter. Data were normalized to percent of binding in vehicle-treated controls with cold GTPγS CPMs subtracted from stimulated CPMs.
Schattauer S.S., Land B.B., Reichard K.L., Abraham A.D., Burgeno L.M., Kuhar J.R., Phillips P.E., Ong S.E, & Chavkin C. (2017). Peroxiredoxin 6 mediates Gαi protein-coupled receptor inactivation by cJun kinase. Nature Communications, 8, 743.
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10

Competitive Binding Assay for Aβ1-42 Aggregates

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The Aβ1–42 aggregation was prepared according
to methods reported previously.31 (link) Competitive
binding assays were carried out
as the procedures below. Borosilicate glass tubes (12 mm × 75
mm) contained aggregated Aβ1–42 aggregation
(100 μL, 0.76 μM), [125I]IMPY (100 μL,
approximately 100 000 cpm), bovine serum albumin solution (700
μL, 0.1% in water), and complex 14–17 (100 μL, 10–4 to 10–9.5 M in ethanol) was incubated at 37 °C for 2 h. Then, the mixture
was filtered by Whatman GF/B filters on a Brandel Mp-48T cell harvester
for separating the bound and free radioactivity. The filter sections
with bound [125I]IMPY were counted using a γ-counter.
After repeating three times, the half maximal inhibitory concentrations
(IC50) were calculated using GraphPad Prism 4.0, and the
inhibition constant (Ki) values were calculated
with by the Cheng–Prusoff equation: Ki = IC50/(1 + [L]/Kd).32 (link)
Song J., Peng X., Li L., Yang F., Zhang X., Zhang J., Dai J, & Cui M. (2018). Al18F-NODA Benzothiazole Derivatives as Imaging Agents for Cerebrovascular Amyloid in Cerebral Amyloid Angiopathy. ACS Omega, 3(10), 13089-13096.
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