LSD1 enzymatic activity assays were performed in triplicate using Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen) with recombinant LSD1 and a synthetic peptide corresponding to the first 21 amino acids of H3K4me2 (Anaspec). LSD1 (500 ng per well) was incubated with 20 μM peptide at room temperature for 30 min. The end point fluorescence was measured on a microplate reader (excitation: 530 nm; emission: 590 nm) after 60 min following the addition of the Amplex Red/HRP mixture. Inhibition assays were performed as described above. In brief, LSD1 (75 ng per well) and inhibitors at the appropriate concentration were incubated at room temperature for 10 min in reaction buffer with 0.01% BRIJ35 (Thermo Fisher Scientific) before the addition of peptide. Ki values were determined in GraphPad Prism v.7 by nonlinear regression analysis (one site-fit Ki) of the concentration/inhibition data.
Brij 35
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Brij-35 is a non-ionic surfactant commonly used in various laboratory applications. It functions as a detergent to solubilize and disperse biological and chemical samples.
Automatically generated - may contain errors
Lab products found in correlation
Neutravidin, by Thermo Fisher Scientific (3 mentions)
Pluronic f 127, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Igg cocktail, by Roche (2 mentions)
Slide a lyzer, by Thermo Fisher Scientific (2 mentions)
Ez link sulfo nhs lc biotin kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Sodium acetate trihydrate, by Merck Group (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Gelatin, by Thermo Fisher Scientific (2 mentions)
Phastgel blue r tablets, by GE Healthcare (2 mentions)
Tetrahydrofuran, by Thermo Fisher Scientific (2 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (2 mentions)
Deuterium oxide, by Thermo Fisher Scientific (2 mentions)
Myristyl trimethylammonium bromide, by Merck Group (2 mentions)
Ethyl α bromoisobutyrate, by Merck Group (2 mentions)
Bis 2methacryloyl oxyethyl disulfide, by Merck Group (2 mentions)
Copper 2 bromide, by Thermo Fisher Scientific (2 mentions)
Tris 2 carboxylethyl phosphine tcep, by Merck Group (2 mentions)
Tert butyl methacrylate, by Merck Group (2 mentions)
39 protocols using brij 35
1
LSD1 Activity and Inhibition Assay
2
Quantitative Amino Acid Analysis
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Amino acid standards, sodium acetate trihydrate, hydrochloric acid, 2-mercaptoethanol, HPLC grade methanol and acetonitrile were obtained from Sigma Aldrich (St. Louis, MO, USA). β-alanine and OPA were purchased from TCI Chemicals (Portland, OR, USA). l -ornithine, glacial acetic acid, Brij-35 and sodium borate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HPLC-grade water (resistivity 18.2 mΩcm) was obtained from a Nanopure water purification system (Barnstead, Dubuque, IA).
3
Quantifying Anti-Pneumococcal IgG Titers
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Anti-pneumococcal IgG titers were assessed in serum from uninfected and infected mice using whole bacterial cells as the capture antigen, as described previously with some adjustments [28 (link)]. Type 23F strain was grown to an OD620 of 1 and washed with PBS. Pneumococcal cells were diluted to final OD620 of 0.1 in coating buffer (0.015 M Na2CO3, 0.035 M NaHCO3). Microtiter plates (2HB, Immunolon) were coated with 100 μL suspension/well at 4°C overnight. The next day, the plates were blocked with 1% bovine serum albumin (BSA) in PBS at room temperature for 1 hour, after which the plates were incubated with serial serum dilutions in PBS at 4°C overnight. Peroxidase-conjugated goat anti-mouse IgG (Jackson Immuno Research Laboratories) was applied and plates were incubated at 37°C for 1.5h. Between incubation steps, plates were washed three times with 0.05% Brij-35 (Thermo Fisher Scientific) in PBS. Plates were developed using 100 μL/well substrate o-phenylenediamine dihydroxychloride (OPD, Thermo Fisher Scientific, 1 tablet in 7.5 ml H2O with 7.5 μL 30% H2O2) and incubated at room temperature for 30 minutes in the dark. Reactions were stopped with 50 μl/well of 2M H2SO4 and the absorbance was measured at 492 nm. Serum IgG titers were determined by calculating the dilution at which the absorbance was equal to an OD492 of 0.1.
4
Affinity Purification of APC Protein
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Cold APC purification buffer [100 mM NaPi, Sigma #S3139 and Sigma #S9390, 300 mM KCl, Sigma #P9333, 5 mM MgCl2 × 6 H2O, Sigma #M2670, 0.001% Brij35 Thermo Fisher Scientific, #28316, 2.5 mM dithiothreitol (DTT), Sigma #D0632, 2.5 mM EDTA, Sigma #EDS] supplemented with protease inhibitors (Roche, #5056489001) and deoxyribonuclease I (DNaseI) (Roche, #10104159001) was added to frozen SF21 cell pellets expressing an APC construct. The pellet was thawed in a room temperature water bath and resuspended. The lysate was clarified by centrifugation (184.000g, 45 min, 4°C), and the supernatant was applied to a 5-ml HiTrap immunoglobulin G Sepharose FF column (GE Healthcare, #28-9083-66). After washing with APC purification buffer, GST (glutathione S-transferase)–TEV protease was added to the column, and the column was left at 4°C overnight to cleave APC off its column-bound affinity and solubility tags. The next day, APC was eluted with APC purification buffer and labeled with SNAP-reactive dye (NEB #S9105S). After SNAP labeling, APC was concentrated using Vivaspin concentrators (Sartorius), ultracentrifuged (280,000g, 10 min, 4°C), and gel filtered using a Superose6 10/300 GL column (GE Healthcare). Peak fractions were pooled, and the labeling ratio was determined using NanoDrop (Thermo Fisher Scientific). The protein was aliquoted on ice and snap frozen in liquid nitrogen.
5
Optimized Microarray Assay Buffers
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The buffers and biological solutions used in the microarray assays included the following: 1X phosphate-buffered saline (PBS) + 0.5% or 0.1% Tween-20 (PBST 0.5 or 0.1 ); 10× sample buffer (1× PBS + 1% Tween-20 + 1% Brij-35; Thermo Scientific, Rockford, IL); 4× IgG blocking cocktail (400 μg/mL each of mouse, sheep, and goat IgG, 800 μg/mL rabbit IgG in 1× PBS, antibodies from Jackson Immunoresearch, West Grove, PA); 10× protease inhibitor (Complete Tablet; Roche Applied Science, Indianapolis, IN); and 2× sample dilution buffer (2× sample buffer + 2× protease inhibitor + 2× IgG cocktail in 1× PBS).
The antibodies and lectins were acquired from various sources (Supplementary Table 2 ). The capture antibodies to be printed onto microarray slides were purified by dialysis (Slide-A-Lyzer; Pierce Biotechnology, Rockford, IL) to 1× PBS and ultracentrifuged. Biotinylation was performed using the EZ-Link-sulfo-NHS-LC-Biotin kit (Pierce Biotechnology) according to the manufacturer’s instructions.
The antibodies and lectins were acquired from various sources (
6
Vi PS Capture ELISA for Typhoid Vaccine Identification
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A Vi PS capture ELISA was performed at NIBSC to determine the identity of Vi PS in typhoid vaccines, according to Hitri et al. [15 (link)] with some modifications. Following the coating of plates (Nunc Maxisorp) with horse anti-mouse IgG and a blocking step with 1% w/v BSA, TCV and Vi PS vaccine samples were diluted from 1:100 to 1:102,400 in assay buffer (PBS with 0.1% v/v Brij-35 (Thermo Scientific, Waltham, MA, USA 20150) and 1% w/v BSA) in 2-fold dilutions across the plate, with a final volume of 100 µL. Plates were incubated at room temperature for 1 h, washed, and 100 µL of rabbit anti-Vi serum (NIBSC 04/152) diluted 1:5000 in assay buffer was added to the wells and incubated at room temperature for 2 h. Plates were washed, and bound IgG was detected by incubation with 100 µL goat anti-rabbit IgG-HRP (Sigma, Kawasaki City, Japan) diluted 1:10,000 in assay buffer per well at room temperature for 1 h. Plates were developed, and ODs were read at 450 nm. CombiStats software was used to evaluate the binding curves. The sample identity is positive if the Vi PS content is calculated to be NLT 40 µg/mL based on the dose–response curve of the reference Vi PS preparation (NIBSC 16/126), and the curve should be comparable with NIBSC 16/126 with no significant deviations from parallelism or linearity.
7
Microtubule Immobilization for Microscopy
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The assay was performed following a previously described protocol with some changes (Roostalu et al., 2015 (link)). Flow chambers were incubated with 5% Pluronic F-127 in water (Invitrogen, P6866) for 10 min at room temperature and then washed with assay buffer (80 mM Pipes, 30 mM KCl, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP, 5 mM 2-mercaptoethanol, 0.075% (w/v) methylcellulose (4000 cP; Sigma-Aldrich, M0512), 1% (w/v) glucose, 0.02% (v/v) Brij-35 (Thermo Scientific, 20150)) supplemented with 50 μg mL−1 k-casein (Sigma-Aldrich, C0406) and extra 0.012% (v/v) Brij-35. Flow chambers were then incubated with assay buffer containing 50 μg mL−1 NeutrAvidin (Invitrogen, A2666) for 3 min on a metal block on ice and subsequently washed with BRB80 (80 mM Pipes, 1 mM EGTA, 1 mM MgCl2). Next, flow chambers were incubated for 5 min at room temperature with biotin- and Alexa-568-labeled GMPCPP-stabilized microtubules diluted 1:2000 in BRB80. Unbound microtubules were removed by subsequent BRB80 washes.
8
Urea Determination Protocol via Colorimetric Assay
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For urea determination, an assay according to Zawada et al. was established [35 (link)]. The following reagents were used. O-Phthalaldehyde reagent: 80 mL dH2O were mixed carefully with 7.4 mL 2.5 mol/L H2SO4 on ice. 60 mg o-Phthalaldehyde and 100 µL 30% Brij-35 (Thermo Scientific) were added to the cooled solution and diluted with dH2O to 100 mL. Primaquine reagent: 500 mg boric acid was dissolved in 60 mL dH2O and mixed carefully with 22.2 mL 2.5 mol/L H2SO4 on ice. 102.6 mg Primaquine and 100 µL 30% Brij-35 were added to the cooled solution and diluted with dH2O to 100 mL. For the urea assay, 50 µL of sample was mixed with 100 µL of O-Phthalaldehyde and shaken gently. Afterwards, 100 µL of primaquine reagent was added and incubated for 1 h at 37 °C. Absorbance was measured at λ = 430 nm by using a microplate photometer.
9
Preparing Microtubule Assay Chambers
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Flow chambers were incubated with 5% Pluronic F-127 in water (Invitrogen, P6866) for 5 min at room temperature and then washed with assay buffer (BRB80, 5 mM β-mercaptoethanol, 0.075% (w/v) methylcellulose, 1% (w/v) glucose, 0.02% (v/v) Brij-35 (Thermo Scientific, 20150)) supplemented with 50 μg/mL κ-casein. Flow chambers were then incubated with an assay buffer containing 50 μg/mL NeutrAvidin (Invitrogen, A2666) for 2 min on a metal block on ice and subsequently washed with BRB80. Next, flow chambers were incubated for 5 min at room temperature with Alexa-568 labeled, biotinylated GMPCPP-stabilized MTs diluted 1:2000 in BRB80. Unbound MTs were removed by additional BRB80 washes.
10
Gelatin Zymography Analysis of MMP-9 Regulation by ApoE Isoforms
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