The ability of plasma to inhibit recombinant HDAC1 was assessed with HDAC1 inhibitor screening assay kit (Cayman Chemical, Ann Arbor, MI) following manufacturer instructions. Plasma was tested at 10 μl, 20 μl, and 40 μl in a total reaction volume of 170 μl. Each reaction included 200 μM substrate (Km = 100 μM). The data are presented as percent initial activity. The signal from wells lacking HDAC1 were defined as the background signal and subtracted from the signal of each sample. This was divided by the activity of untreated HDAC1 to calculate the percent of initial activity.
Hdac1 inhibitor screening assay kit
Manufactured by Cayman Chemical
Sourced in United States
The HDAC1 Inhibitor Screening Assay Kit is a laboratory tool used to measure the inhibitory activity of compounds against the HDAC1 enzyme. The kit provides the necessary components to perform in vitro screening of potential HDAC1 inhibitors.
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Lab products found in correlation
Microplate reader, by Tecan (1 mentions)
Valproic acid, by Fujifilm (1 mentions)
Hrp conjugated anti mouse igg antibody, by Merck Group (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Anti mouse igg peroxidase antibody, by Merck Group (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated anti rabbit igg antibody, by Merck Group (1 mentions)
Anti yy1 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti nf κb, by Cell Signaling Technology (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Anti phospho stat1, by Cell Signaling Technology (1 mentions)
Anti phospho stat3, by Cell Signaling Technology (1 mentions)
Spectramax id3, by Molecular Devices (1 mentions)
5 protocols using hdac1 inhibitor screening assay kit
1
Plasma HDAC1 Inhibition Screening Assay
2
HDAC1 Inhibitor Screening Assay
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HDAC activity was determined using HDAC1 Inhibitor Screening Assay Kit (Cayman Chemical). After adding 140 μl of assay buffer, 10 μl of recombinant HDAC1, and 10 μl of each sample to a 96-well plate, 10 μl of HDAC substrate was added and the plate was incubated at 37°C for 30 min. Then, 40 μl of HDAC developer was added, and the plate was incubated at room temperature for 15 min, followed by measuring the fluorescence (Ex 365 nm, Em 410–460 nm). Each HDAC activity is expressed as a percentage relative to the control.
3
Evaluating HDAC Inhibitory Potential
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The HDAC inhibitory potential of the novel compounds was determined by utilizing the deacetylase activity of recombinant human HDAC1 (HDAC1 Inhibitor Screening Assay Kit; Cayman Chemicals) or HDAC6 (HDAC6 Inhibitor Screening Kit (Fluorometric); Biovision) towards the corresponding synthetic acetylated-peptide substrates resulting in the release of a fluorescent product. The assays were performed according to manufacturer’s description of the in commercially available assay kits. The fluorescence intensity (HDAC1: λex = 352 nm, λem = 452 nm; HDAC6: λex = 380 nm, λem = 510 nm) as a measure of enzyme activity was measured at 37 °C with a microplate reader (Tecan). The IC50 values were derived from dose-response curves and are expressed as the means ± SD of two independent experiments. A two-tailed t-test was performed, revealing significant differences (p < 0.0001) in HDAC6-inhibition for 4e –f compared with 4d as well as for 4d and 4f compared with 4e and in HDAC1-inhibition for 4d –e compared with 4f . The difference in HDAC1 IC50 values of 4d compared with 4e were not significant.
4
Cellular Signaling Modulation Protocol
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Sebacic acid, valproic acid, and O-tetradecanoylphorbol-13-acetate (TPA) were purchased from FUJIFILM Wako Pure Chemicals (Osaka, Japan). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and anti-mouse IgG peroxidase antibody were purchased from Sigma-Aldrich (St. Louis, MO). Brefeldin A was purchased from Tokyo Chemical Industry (Tokyo, Japan). Anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, anti-NF-κB, anti-phospho-STAT1, anti-STAT1, anti-phospho-STAT3, and anti-STAT3 antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-YY-1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody and HRP-conjugated anti-mouse IgG antibody were purchased from Sigma-Aldrich. HDAC1 Inhibitor Screening Assay Kit was purchased from Cayman Chemical (Ann Arbor, MI).
5
HDAC1 Inhibitor Screening Assay
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The activity of HDAC1 was measured using HDAC1 Inhibitor Screening Assay Kit (Cayman Chemical, Ann Arbor, MI, U.S.A.), according to manufacturer's instructions. For this analysis, we serially diluted in the HDAC1 Inhibitor Screening assay buffer a commercial liquid lacosamide preparation (Vimpat solution for Intravenous use; UCB, Inc., Brussels, Belgium). Human recombinant HDAC1 was incubated in a 96‐well microplate with 200 μm acetylated substrate in assay buffer containing or lacking lacosamide (total volume 170 μl). An additional negative control was incubation of the substrate without HDAC1. Trichostatin A (TSA) at 12.35 μm and valproic acid at the indicated concentrations served as the positive controls. Following incubation at 37°C for 30 minutes, the reaction was stopped by addition of 40 μl TSA‐containing developer. The plate was incubated for an additional 15 minutes at room temperature, and the newly formed fluorophore was detected on a fluorometric reader (SpectraMax iD3; Molecular Devices, San Jose, CA, U.S.A.; excitation at 350 nm, emission at 450 nm). Fluorescence was measured by an independent lab and the raw data were translated to percent HDAC inhibition as per the kit manufacturer's instructions. Results are reported as percentage HDAC1 activity as compared to control (activity in the absence of inhibitors).
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