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Tri carb 2900tr liquid scintillation analyzer

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The Tri-Carb 2900TR Liquid Scintillation Analyzer is a laboratory instrument designed for the detection and measurement of radioactive samples. It utilizes liquid scintillation counting technology to quantify the radioactivity present in liquid samples.

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Lab products found in correlation

Net434250uc, by PerkinElmer (2 mentions) Amherst protran 0.1 μm nitrocellulose membrane, by Cytiva (2 mentions) 14c ac coa, by PerkinElmer (2 mentions) P81 phosphocellulose filter discs, by Merck Group (2 mentions) Ultima gold f scintillation mixture, by PerkinElmer (2 mentions) Ultima gold f, by PerkinElmer (1 mentions) 3h corticosterone, by PerkinElmer (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Rneasy plus mini kit, by Qiagen (1 mentions)

13 protocols using tri carb 2900tr liquid scintillation analyzer

1

Micellar Detergent-Based Sphingomyelin Hydrolysis Assay

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The micellar detergent-based assay was performed as previously described (10 (link), 35 (link)).
Assay mixtures contained 250 mM sodium-acetate buffer (pH 4.5), 0.1% (w/v) Nonidet P-40, 3.8 ng purified ASM, and a mixture of unlabeled SM and [3H]SM (labeled at the methyl residue of the choline group) with a final SM concentration of 100 μM/50 μl assay volume. The assay was incubated at 37°C for a time period of 0–120 min. The reactions were terminated by adding 800 μl chloroform/methanol 2/1 (v/v) and 250 μl water. The upper aqueous layer was separated from the organic layer by gentle vortexing and centrifugation at 12,000 g. Liberated [3H]phosphorylcholine was quantified in a 200 μl aliquot from the upper phase using a Tricarb 2900TR liquid scintillation analyzer (PerkinElmer, Rodgau, Germany). One unit of enzymatic activity is defined as the amount of enzyme that catalyzes the hydrolysis of 1 mmol SM per hour in the detergent-based assay system.
Oninla V.O., Breiden B., Babalola J.O, & Sandhoff K. (2014). Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2. Journal of Lipid Research, 55(12), 2606-2619.
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2

OGT-Mediated CKII Peptide O-GlcNAcylation

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Purified OGT (500 nM of wild-type or N791A mutant) was incubated with 50 μM UDP-3H-GlcNAc (specific activity 0.3 Ci/mmol, PerkinElmer #NET434250UC) and 500 μM CKII peptide (YPGGSTPVSSANMM) in the reaction buffer (20 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM THP) at 37°C for 3 hours. Reactions were quenched by spotting the samples onto Amherst Protran 0.1 μm nitrocellulose membrane (Cytiva), air-dried, and washed for 5 min four times in PBS buffer. The radioactivity of each membrane was counted by a Tri-Carb 2900 TR Liquid Scintillation Analyzer (PerkinElmer). A reaction without OGT was set up as negative control. Another reaction without the washing steps was counted as a total of 50 μM of UDP-GlcNAc input to calculate the O-GlcNAcylation level of CKII peptide in each reaction. The experiment was performed in triplicate.
Blankenship C., Xie J., Benz C., Wang A., Ivarsson Y, & Jiang J. (2023). A novel binding site on the cryptic intervening domain is a motif-dependent regulator of O-GlcNAc transferase. Research Square.
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3

GPAT1 Activity Assay in Mitochondria

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GPAT1 activity assay was performed according to the previously reported procedure [27 (link)]. NEM or H2O2 was added to the reaction mixture containing 75 mM Tris-HCl (pH 7.5), 4 mM MgCl2, 8 mM NaF, 100 μM palmitoyl-CoA, 1.8 μM [14C] glycerol-3-phosphate and 2 mg/ml BSA. The reaction was started by adding 2 μg of mitochondrial membrane fraction to the reaction mixture in a final volume of 200 μl and then incubated at 26°C for 20 min. The reaction was stopped by mixing with 1 ml of water-saturated butanol. [14C] lysophosphatidic acid (LPA) was extracted by mixing the mixture with 0.5 ml of butanol-saturated water, and 0.8 ml of the organic phase was collected from it. Thereafter, it was washed once with 0.8 ml of butanol-saturated water. The radioactivity of the top phase was determined by liquid scintillation counting (Tri-Carb 2900TR Liquid Scintillation Analyzer, PerkinElmer).
Jung S., Choi M., Choi K., Kwon E.B., Kang M., Kim D.E., Jeong H., Kim J., Kim J.H., Kim M.O., Han S.B, & Cho S. (2017). Inactivation of human DGAT2 by oxidative stress on cysteine residues. PLoS ONE, 12(7), e0181076.
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4

Nt-Acetylation Activity Assay of NAA10

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To compare the intrinsic enzyme activity of V5-immunoprecipitated NAA10 WT-V5 and NAA10 N101K-V5, Nt-acetylation assays were performed as described previously [37 ]. In short, we used triplicate reactions containing 10 µl immunoprecipitated enzyme, 50 µM [14C]-Ac-CoA (PerkinElmer, MA, USA), 200 µM oligopeptide SESS24 (SESSSKSRWGRPVGRRRRPVRVYP) or EEEI24 (EEEIAALRWGRPVGRRRRPVRVYP) (BioGenes, Berlin, Germany), and acetylation buffer with a total volume of 25 µl. Oligopeptide was omitted in negative control reactions. Reactions were run for 30 min at 37 °C and 1400 rpm in a thermomixer. Afterwards, 23 µl of the supernatant was transferred onto P81 phosphocellulose filter discs (Millipore, MA, USA) which were subsequently washed three times in 10 mM HEPES buffer (pH 7.4) and air dried. Finally, the filter discs were submerged in 5 ml Ultima Gold F scintillation mixture (PerkinElmer, MA, USA) and the Nt-acetylated product was measured by a TriCarb 2900TR Liquid Scintillation Analyzer (PerkinElmer, MA, USA).
McTiernan N., Gill H., Prada C.E., Pachajoa H., Lores J, & Arnesen T. (2020). NAA10 p.(N101K) disrupts N-terminal acetyltransferase complex NatA and is associated with developmental delay and hemihypertrophy. European Journal of Human Genetics, 29(2), 280-288.
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5

Assaying DGAT Activities in Membranes

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DGAT activities in total membranes, which were prepared from human DGAT2- or DGAT1-overexpressing Sf9 cells, were determined by measuring the formation of [14C] TG from [14C] oleoyl-CoA and sn-1,2-diacylglycerol as described previously [27 (link)]. The reaction mixture for DGAT2 assay contains 175 mM Tris-HCl (pH 7.5), 5 mM MgCl2 (100 mM MgCl2 for DGAT1 assay), 200 μM sn-1,2-diacylglycerol, 20 μM [14C] oleoly-CoA (5.5 μCi), 2 mg/ml BSA and 32 μg (50 μg for DGAT1 assay) of the membrane protein. The mixture was incubated with thiol-modifying reagents in the presence or absence of 20 mM DTT at 37°C for 20 min. Thereafter, the reaction was stopped by the addition of 1.5 ml of stop solution [2-propanol/heptane/water (80:20:2, v/v/v)] and vortexed with 1 ml of heptane and 0.5 ml of water. The top heptane phase was collected and washed with 2 ml alkaline ethanol solution [ethanol/0.5 N NaOH/water (50:10:40, v/v/v)]. The radioactivity of the top phase was quantified by liquid scintillation counting (Tri-Carb 2900TR Liquid Scintillation Analyzer, PerkinElmer).
Jung S., Choi M., Choi K., Kwon E.B., Kang M., Kim D.E., Jeong H., Kim J., Kim J.H., Kim M.O., Han S.B, & Cho S. (2017). Inactivation of human DGAT2 by oxidative stress on cysteine residues. PLoS ONE, 12(7), e0181076.
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6

Measurement of Arachidonic Acid Release

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To measure the Arachidonic acid (AA) release, we used the pulse-chase metabolic chasing method according to the published protocol [16 (link)]. The AA [5 (link),6 (link),8 (link),9 (link),11 (link),12 (link),14 ,15 (link)-3H(N)] was purchased from PerkinElmer. Briefly, BMDMs were plated at a density of 0.5 ×106 million/per well in 6-well plate and incubated overnight at 37 °C in a humidified incubator. The cells were pretreated with or without 1 μM of γT3 for 12 hours, then incubated [3H]-AA (0.5 μCi/1mL/well) for 18 h at 37 °C for loading. The cells were washed three times with 2 mg/mL of bovine serum albumin in phosphate-buffered saline to remove unincorporated [3H]-AA, followed by NLRP3 inflammasome stimulation using LPS/palmitate as described in 2.3. After 6 hours of NLRP3 inflammasome activation, released [3H]-AA to the culture medium was collected, centrifuged to remove cell debris, and used for measurement of [3H] radioactivity by liquid scintillation counter (Tri-carb 2900TR liquid scintillation analyzer, Perkin Elmer).
Kim Y., Gromovsky A.D., Brown J.M, & Chung S. (2018). Gamma-tocotrienol attenuates the aberrant lipid mediator production in NLRP3 inflammasome-stimulated macrophages. The Journal of nutritional biochemistry, 58, 169-177.
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7

Quantitative Peptide Acetylation Assay

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The peptide acetylation assay was performed as we have described (7 (link), 29 (link)). Briefly, NAA80 (E. coli–expressed or immunoprecipitated from HeLa cells) was mixed with 300 μM synthetic peptides (synthesized and quality-controlled by BioGenes; fig. S9) and 50 μM isotope-labeled [14C]-AcCoA (PerkinElmer) in a total volume of 25 μl of acetylation buffer (see above). The samples were incubated for 1 hour at 37°C, and 20 μl was then transferred onto P81 phosphocellulose filter discs (Millipore). The discs were washed three times with 10 mM Hepes (pH 7.4) and dried before they were added to 5 ml of scintillation cocktail Ultima Gold F (PerkinElmer). The amount of incorporated [14C]-Ac was determined by a Tri-Carb 2900TR Liquid Scintillation Analyzer (PerkinElmer). The activity was corrected for the amount of immunoprecipitated NAA80 determined by Western blot analysis.
Rebowski G., Boczkowska M., Drazic A., Ree R., Goris M., Arnesen T, & Dominguez R. (2020). Mechanism of actin N-terminal acetylation. Science Advances, 6(15), eaay8793.
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8

Acetylation Assay for Peptide Modification

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Three positive replicates were prepared for each IP sample containing 10 µL IP beads, 200 µM synthetic oligopeptide SESS24 (BioGenes), 100 µM [14C]-Ac-CoA (Perkin-Elmer), and dH2O to a final volume of 25 µL. In addition, two replicates for each IP sample were prepared without synthetic oligopeptide as negative controls. The samples were incubated at 37°C for 45 min in a thermomixer with shaking at 1400 rpm. Finally, the magnetic beads were isolated and 23 µL of the supernatant transferred to P81 phosphocellulose filter disks (Millipore). The filter disks were washed three times for 5 min in 10 mM HEPES buffer (pH 7.4) and air dried. To determine the amount of incorporated [14C]-Ac, the filter disks were added to 5 mL Ultima Gold F scintillation mixture (Perkin-Elmer) and analyzed by a Perkin-Elmer TriCarb 2900TR Liquid Scintillation Analyzer.
Kweon H.Y., Lee M.N., Dorfel M., Seo S., Gottlieb L., PaPazyan T., McTiernan N., Ree R., Bolton D., Garcia A., Flory M., Crain J., Sebold A., Lyons S., Ismail A., Marchi E., Sonn S.K., Jeong S.J., Jeon S., Ju S., Conway S.J., Kim T., Kim H.S., Lee C., Roh T.Y., Arnesen T., Marmorstein R., Oh G.T, & Lyon G.J. (2021). Naa12 compensates for Naa10 in mice in the amino-terminal acetylation pathway. eLife, 10, e65952.
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9

Radioimmunoassay for Corticosterone Quantification

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CORT was measured using RIA as previously described27 (limit of detection = 4.45 ng/ml). Plasma samples were diluted 1:25 in 0.01 M phosphate buffered saline (PBS) (pH = 7.4). CORT binding globulin was denatured by heating samples at 65C for 1 h. A standard curve ranging from 2.5 pg to 750 pg CORT (Steraloids) was run alongside all assay tubes. All samples and standards were incubated with CORT antiserum (1:1200, Cat# 7120016, RRID:AB_2801269; MP Biomedicals) and 3H corticosterone (Perkin Elmer) overnight at 4°C. Unbound CORT was removed by adding dextran‐coated charcoal and centrifuging for 15 min at 3000 rpm and 4°C. The supernatant containing bound CORT was decanted into plastic scintillation vials, mixed with 4 ml liquid scintillation cocktail (Ecoscint Ultra; National Diagnostics), and counted using a Tri‐Carb 2900TR (Packard Tri‐Carb 2900TR Liquid Scintillation Analyzer, RRID:SCR_018610; PerkinElmer). Sample CORT concentrations were determined via comparison to a standard curve (2.5‐750 pg) and data was analyzed using Graphpad Prism Software (GraphPad Prism, RRID:SCR_002798). The intra‐assay variance was 9.4% and was determined using internal quality controls measured throughout the assay. All samples from a single experiment were run in a single assay.
Miller A.M., Daniels R.M., Sheng J.A., Wu T.J, & Handa R.J. (2022). Glucocorticoid regulation of diurnal spine plasticity in the murine ventromedial prefrontal cortex. Journal of Neuroendocrinology, 34(12), e13212.
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10

O-GlcNAc Transferase Kinetic Assay

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Purified OGT (500 nM of wild-type or N791A mutant) was incubated with 50 μM UDP-3 H-GlcNAc (specific activity 0.3 Ci/mmol, PerkinElmer #NET434250UC) and 500 μM CKII peptide (YPGGSTPVSSANMM) in the reaction buffer (20 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM THP) at 37°C for 3 hours. Reactions were quenched by spotting the samples onto Amherst Protran 0.1 μm nitrocellulose membrane (Cytiva), air-dried, and washed for 5 min four times in PBS buffer. The radioactivity of each membrane was counted by a Tri-Carb 2900 TR Liquid Scintillation Analyzer (PerkinElmer). A reaction without OGT was set up as negative control. Another reaction without the washing steps was counted as a total of 50 μM of UDP-GlcNAc input to calculate the O-GlcNAcylation level of CKII peptide in each reaction. The experiment was performed in triplicate.
Blankenship C.M., Xie J., Benz C., Wang A., Ivarsson Y, & Jiang J. (2023). Motif-dependent binding on the intervening domain regulates O-GlcNAc transferase. Nature chemical biology, 19(11).
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