His-DNMT1 (182 kDa, human) was cloned, expressed and purified as described by Lee et al. [20] (link). The DNMT1 assay was performed according to Gros et al. [21] (link). Briefly, the reaction was started by addition of 90 nM of DNMT1 on a mix containing the tested compound (up to 1% DMSO), 1 µM of a AdoMet/[methyl-3H]-AdoMet mix in a ratio of 3-to-1 (isotopic dilution 1*:3) and 0.3 µM of biotinylated DNA duplex in 10 µL final volume. The reaction was incubated at 37°C for 2 h in reaction buffer (20 mM HEPES pH 7.2, 1 mM EDTA, 50 mM KCl, 25 µg/mL BSA). 8 µL are then transferred into a streptavidin-coated Flashplate PLUS (PerkinElmer) containing 190 µL of 20 µM AdoHcy (Sigma-Aldrich) in 50 mM Tris-HCl pH 7.4. The Flashplate was agitated at room temperature for 1 h, washed three times with 200 µL of 0.05% Tween-20 in 50 mM Tris-HCl pH 7.4 and read in 200 µL of 50 mM Tris-HCl pH 7.4 on TopCount NXT (PerkinElmer). Percentages of inhibition were calculated with the same formula as in DNMT3A assay.
Flashplate plus
Manufactured by PerkinElmer
The Flashplate PLUS is a lab instrument designed for high-throughput screening and detection of radioactive samples. It provides a reliable and efficient platform for processing and analyzing multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Topcount nxt, by Hewlett-Packard (2 mentions)
3h sam, by PerkinElmer (2 mentions)
Topcount nxt microplate scintillation and luminescence counter, by PerkinElmer (2 mentions)
Topcount nxt hts, by PerkinElmer (2 mentions)
Adohcy, by Merck Group (1 mentions)
Topcount nxt, by PerkinElmer (1 mentions)
Topcount reader, by PerkinElmer (1 mentions)
9 protocols using flashplate plus
1
DNMT1 Enzymatic Assay Protocol
2
Peptide-HLA Stability Measurement by SPA
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The stability of peptide-HLA class I complexes was measured using dissociation of 125I radiolabelled β2m in a scintillation proximity assay (SPA) as previously described (69 (link)). Briefly, recombinant, biotinylated HLA class I heavy chains were diluted into a refolding buffer containing the test peptide and trace amounts of 125I radiolabeled β2m, and allowed to refold at 18°C for 24 h in a Streptavidin-coated scintillation microplate (Flashplate PLUS, Perkin Elmer, Boston, MA). Dissociation was initiated by adding excess unlabeled β2m and placing the microplate in a scintillation counter (TopCount NXT, Packard) adjusted to 37°C. Reading the microplate continuously for 24 h allowed determination of the dissociation of radiolabeled β2m.
3
Measuring Peptide-HLA Stability Using Radiolabel
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The stability of peptide-HLA class I complexes was measured using 125I radiolabelled β2m in a scintillation proximity assay as previously described [46] (link). Briefly, recombinant, biotinylated HLA class I heavy chains were diluted into a refolding buffer containing the test peptide and trace amounts of 125I radiolabeled β2m, and allowed to refold at 18°C for 24 h in a Streptavidin-coated scintillation microplate (Flashplate PLUS, Perkin Elmer, Boston, MA). Dissociation was initiated by adding excess of unlabeled β2m and placing the microplate in a scintillation counter (TopCount NXT, Packard) adjusted to 37°C. The microplate was read continuously for 24 h thereby allowing the dissociation of radiolabeled β2m to be determined.
4
PRMT Inhibitor Screening Using Scintillation Proximity Assay
5
Scintillation Proximity Assay for Methyltransferase Inhibitor Screening
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The assay conditions and protein constructs used in this study are presented in Supplementary Tables 4 and 5 , respectively. The protein purification procedures have been previously described [17 (link), 23 (link), 29 (link), 30 (link)]. In the scintillation proximity assay (SPA), specific amounts of enzyme and biotinylated peptide substrate were mixed with the compound and the reaction was initiated by adding SAM. IC50 values were measured at the apparent Km concentrations of the substrate and SAM. The reaction was quenched by adding an equal volume of 7.5 M guanidine hydrochloride, and the reaction product was measured by SPA using FlashPlate Plus and TopCount NXT HTS plate reader (both from Perkin Elmer Life Sciences).
6
Inhibitory Effects of Compounds on PRMT Enzymes
7
Optimizing PRDM9 Methylation Assay
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The radioactivity-based assay described above was used to determine the best assay conditions for the PRDM9 methylation reaction. For the pH titration, the reaction mixture contained 10 μm biotinylated peptide and 40 μm AdoMet (2.5 μm [3H]AdoMet plus 37.5 μm cold AdoMet) in 20 mm Bis-tris propane buffer, pH 6.0–9.5, in a final volume of 20 μl. For the NaCl, DMSO, Triton X-100, DTT, and Tris(2-carboxyethyl)phosphine (TCEP) titrations, the reaction mixture contained 10 μm biotinylated peptide and 40 μm AdoMet (2.5 μm [3H]AdoMet plus 37.5 μm cold AdoMet) in 10 mm Tris-HCl, pH 8.5, in a final volume of 20 μl. The reactions were started by adding 100 nm PRDM9. Samples were incubated for 1 h at 23 °C and reactions were quenched by addition of 20 μl of 7.5 m guanidinium hydrochloride followed by addition of 160 μl of 20 mm Tris-HCl buffer, pH 8.0. Samples were added to wells of a streptavidin/scintillant-coated microplate (FlashPlate® PLUS: PerkinElmer Life Sciences). The amount of methylated peptide was quantified by tracing the radioactivity (cpm) as measured by a TopCount reader from PerkinElmer.
8
PRDM9 Histone Methylation Assay
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A radioactivity-based assay was used to determine histone peptide substrate specificity of PRDM9. In this assay [3H]AdoMet (PerkinElmer Life Sciences; catalog number NET155V250UC) was used as a methyl donor to methylate biotinylated histone peptide substrates. Reaction mixtures contained 10 μm biotinylated peptide and 200 μm AdoMet (4 μm [3H]AdoMet plus 196 μm cold AdoMet) in 10 mm Tris-HCl buffer, pH 8.5, with 0.01% Triton X-100 and 10 mm DTT in a final volume of 20 μl. The reaction was started by adding PRDM9 (final concentration of 1 nm ). Samples were incubated for 30 min at 23 °C and the reaction was quenched by addition of 20 μl of 7.5 m guanidinium hydrochloride followed by addition of 160 μl of 20 mm Tris-HCl buffer, pH 8.0. Samples were added to wells of a streptavidin/scintillant-coated microplate (FlashPlate® PLUS; PerkinElmer Life Sciences). Biotinylated peptides would be captured in each well through their interaction with streptavidin, thereby bringing any incorporated [3H]methyl in proximity of scintillant. The amount of methylated peptide was quantified by tracing the radioactivity (cpm) as measured by the TopCount NXTTM Microplate Scintillation and Luminescence Counter (PerkinElmer Life Sciences).
9
Substrate Preparation and Demethylase Assay
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The ALKBH5 substrate, m 6 A-ss-RNA (5′-UACACUCGA UCUGG(m 6 A)CUAAAGCUGCUC-3′-biotin) was generated using the METTL3-14 complex to completely monomethylate N 6 A ss-RNA. Of the METTL3-14, 500 nM was incubated with 5 µM of biotin-labeled ss-RNA and 5 µM of 3 H-SAM in 20 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% Triton X-100, 40 U of RNaseOUT/100 µL buffer for 1 h at 23 °C. Then, the reaction mixture was heat shocked at 85 °C for 15 min to denature the methyltransferase METTL3-14 complex, after which the sample was left on ice to cool down. To determine the kinetic parameters, the demethylase assays were performed in 25 nM ALKBH5, 10 µM 2-OG, 100 µM ascorbate, 20 µM Fe (II), and varying concentrations of monomethylated ss-RNA. An equal volume of 7.5 M guanidine-HCl was used to quench ALKBH5 activity after 1 h reaction at 37 °C. Sixty microliters of 20 mM Tris-HCl, pH 8.0, was added into the quenched wells. The quenched reaction mixtures were transferred to a streptavidin/scintillant-coated microplate (FlashPlate Plus, Perkin Elmer Life Sciences), allowed to bind for at least 1 h, and then the decrease of radioactive signals was detected on a Top-Count NXT HTS (Perkin Elmer).
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