0.5 μM full length IL-17A (R&D Systems) was pre-incubated with DMSO or 50 μM a control compound in PBS at room temperature for 30 min, after which 5 μM sulfonyl fluoride probe compound 4 was added to the protein mixture (final sample volume 50 μl) and incubated for 1 h. Click chemistry was then performed at room temperature for 2 h. Briefly, 38.2 μL of 4% SDS in HEPES buffer (pH 7.5) was added to the sample, followed by the addition of 1.9 μL of 4 mM biotin azide (Life Technologies), 2 μL of 50 mM CuSO4, 5.9 μL TBTA in DMSO:tert-BuOH (1:5), and 2 μL of 50 mM TCEP (final concentrations: 1.5% SDS, 75 μM biotin azide, 1 mM CuSO4, 5% tert-BuOH and 1 mM TCEP). 1 mL of 6 M urea in PBS was added to quench the reaction. The mixture was then incubated with 100 μL high-capacity streptavidin agarose (Thermo Fisher Scientific) at room temperature for 2 h with rotation. Beads were centrifuged at 1000 g for 1 minute and washed 3 times with 1 mL 4 M urea in PBS. Proteins were eluted by adding 90 μL of 2× LDS sample buffer (Life Technologies) and heated at 95 °C for 10 minutes. Eluates were separated on a 12% NuPAGE Bis-Tris gel, transferred to a PVDF membrane, and then analyzed by immunoblot (anti-IL17A antibody from R&D Systems).
Biotin azide
Manufactured by Thermo Fisher Scientific
Sourced in United States
Biotin azide is a chemical compound that contains a biotin moiety and an azide functional group. It is a versatile tool used in biochemical and bioanalytical applications, particularly in the field of protein labeling and detection.
Automatically generated - may contain errors
Lab products found in correlation
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (8 mentions)
Thymidine, by Merck Group (6 mentions)
Cuso4, by Merck Group (4 mentions)
Formaldehyde, by Merck Group (3 mentions)
Protease inhibitor, by Roche (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Bioruptor, by Diagenode (2 mentions)
Click chemistry reagents, by Thermo Fisher Scientific (2 mentions)
Nos2 ab, by Merck Group (2 mentions)
Pureproteome protein a magnetic bead system, by Merck Group (2 mentions)
4plus streptavidin hrp label, by Biocare Medical (2 mentions)
Click it homopropargylglycine, by Thermo Fisher Scientific (2 mentions)
Laemelli buffer, by Bio-Rad (2 mentions)
A10044, by Thermo Fisher Scientific (2 mentions)
Click it protein reaction buffer kit, by Thermo Fisher Scientific (2 mentions)
Dynabeabs myone streptavidine c1, by Thermo Fisher Scientific (2 mentions)
Streptavidin beads, by Thermo Fisher Scientific (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Streptavidin coated magnetic beads, by Thermo Fisher Scientific (1 mentions)
34 protocols using biotin azide
1
IL-17A Covalent Labeling and Detection
2
Isolation of Replication Forks Using iPOND
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To perform iPOND, 1.5 × 108 cells were labelled with 10 μM EdU (Life Technologies) and treated with 4 mM HU for 4 h as indicated42 (link). Cells were crosslinked with 1% formaldehyde for 10 min at room temperature, quenched with 0.125 M glycine and washed with PBS. For the conjugation of EdU with biotin azide, cells were permeabilized with 0.25% Triton X-100 in PBS buffer, and incubated in click reaction buffer (10 mM sodium-L -ascorbate, 20 μM biotin azide (Life Technologies) and 2 mM CuSO4) for 2 h at room temperature. Cells were resuspended in lysis buffer (50 mM Tris-HCl (pH 8.0) and 1% SDS) supplemented with protease inhibitors (Roche), and chromatin was solubilized by sonication in a Bioruptor (Diagenode-Pico) at 4 °C for 24 min. After centrifugation, supernatants were incubated for 1 h streptavidin-MyOne C1 beads (Life Technologies). Beads were washed and captured proteins were eluted by boiling beads in 2 × NuPAGE LDS Sample Buffer (Life Technologies) containing 200 mM dithiothreitol for 40 min at 95 °C. Proteins were resolved by electrophoresis using NuPAGE Novex 4–12% Bis-Tris gels and detected by western blot with the indicated antibodies.
3
Detection of Nascent and Stalled DNA Forks
4
Fluorescent Labeling of OMV-associated DNA
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OMV-associated DNA was labeled using the Click-iT EdU kit (Molecular Probes). Briefly, P. aeruginosa bacteria were cultured in tryptic soy broth (TSB; Bacto, BD) containing 1.2 mM of EdU (5-ethynyl-2-deoxuridine), a nucleoside analogue of thymidine, for 4 h on an orbital shaker (225 rpm) at 37 °C. The EdU-labeled OMVs were isolated as described above and added to epithelial cells in 8-well chambers (Ibidi). After the required incubation, cells were washed twice in PBS before fixing in 4% paraformaldehyde (PFA) and permeabilization in 3% Triton-X 100. Click-iT EdU labeling involves a two-step process. The first step involves incubation with 12.5 μM biotin azide (Molecular Probes), during which the azide moiety binds specifically to the alkyne backbone of the EdU molecule in the presence of a copper catalyst. The second step involves incubation with 5 μg/ml streptavidin-conjugated 568 Alexa Fluor® (Molecular Probes) to enable fluorescent detection of the biotin-azide bound to the EdU.
5
iPOND Protocol for Chromatin Analysis
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The iPOND experiment was performed as described elsewhere51 with minor modifications. Briefly, 108 cells were pulse-labelled with 10 μM EdU (Invitrogen) for 10 min. Immediately after the pulse or after a 1 h chase in fresh medium supplemented with 10 μM thymidine (Sigma), cells were crosslinked with 1% formaldehyde (Sigma) in PBS for 15 min at room temperature under gentle agitation. Crosslink was stopped by addition of 125 mM glycine for 5 min. Cells were harvested by scrapping and washed in ice-cold PBS. Pellets were permeabilized in PBS with 0.5% Triton × 100 for 30 min at room temperature. Biotin-azide (Molecular Probes) was conjugated to EdU by click chemistry for 2 h in click reaction buffer (10 mM sodium-L -ascorbate, 10 μM Biotin-azide, 2 mM CuSO4 in PBS). Cells were lyzed in iPOND lysis buffer (10 mM Hepes-NaOH pH 7.9, 100 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1 mM PMSF, 0.2% SDS, 0.1% sarkozyl, antiproteases) and sonicated on a Bioruptor device (30 cycles of 30 s on/30 s off at the highest setting). Solubilized chromatin was retrieved by centrifugation at 16,000g for 10 min and supernatant was further incubated overnight with magnetic streptavidin beads (Dynabeabs MyOne Streptavidine C1, Invitrogen). Beads were washed once in lysis buffer, once in 500 mM NaCl and twice in lysis buffer. Proteins were eluted by boiling in 1 × Laemmli buffer at 95 °C for 30 min.
6
Profiling Nascent DNA Replication
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Cells were irradiated with 25 J m–2 UVC and pulse-labelled with 10 μM 5-ethynyl-2′-deoxyuridine (EdU, Invitrogen) for 10 min. In Supplementary Figure 5D , the UVC dose was reduced to 5 J m–2 and the labelling period was extended to 20 min. iPOND was performed immediately after the pulse or after a chase in fresh medium supplemented with 10 μM thymidine (Sigma) as already described (37 (link)). Briefly, cells were crosslinked with 1% formaldehyde (Sigma), harvested by scrapping and permeabilized in PBS with 0.5% Triton X100. Biotin-azide (Molecular Probes) was conjugated to EdU by click chemistry for 2 h in click reaction buffer (10 mM sodium-l -ascorbate, 10 mM Biotin-azide, 2 mM CuSO4 in PBS). Cells were lysed in iPOND lysis buffer (10 mM HEPES–NaOH pH 7.9, 100 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1 mM PMSF, 0.2% SDS, 0.1% sarkozyl, antiproteases) and sonicated on a Bioruptor device. Solubilized chromatin was retrieved by centrifugation and subjected to streptavidin pull-down overnight (Dynabeabs MyOne Streptavidine C1, Invitrogen). After extensive bead washing, pull-downed proteins were eluted by boiling in 1× Laemmli buffer at 95°C for 30 min.
7
iPOND Technique for DNA Replication Dynamics
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Cells were irradiated with 25 J.m -2 UVC and pulse-labelled with 10 μM EdU (Invitrogen) for 10 min. In Supplementary Figure 4d, the UVC dose was rEdUced to 5 J.m -2 and the labelling period was extended to 20 min. iPOND was performed immediately after the pulse or after a chase in fresh medium supplemented with 10 μM thymidine (Sigma) as already described 38 . Briefly, cells were crosslinked with 1% formaldehyde (Sigma), harvested by scrapping and permeabilized in PBS with 0.5% TritonX100. Biotin-azide (Molecular Probes) was conjugated to EdU by click chemistry for 2 h in click reaction buffer (10 mM sodium-L-ascorbate, 10 mM Biotin-azide, 2 mM CuSO4 in PBS). Cells were lysed in iPOND lysis buffer (10 mM Hepes-NaOH pH 7.9, 100 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1 mM PMSF, 0.2% SDS, 0.1% sarkozyl, antiproteases) and sonicated on a Bioruptor device. Solubilized chromatin was retrieved by centrifugation and subjected to streptavidin pull-down overnight (Dynabeabs preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted October 12, 2020. ; https://doi.org/10.1101/2020.10.12.336107 doi: bioRxiv preprint 25 MyOne Streptavidine C1, Invitrogen). After bead washing, pull-downed proteins were eluted by boiling in 1x Laemmli buffer at 95 °C for 30 min.
The copyright holder for this this version posted October 12, 2020. ; https://doi.org/10.1101/2020.10.12.336107 doi: bioRxiv preprint 25 MyOne Streptavidine C1, Invitrogen). After bead washing, pull-downed proteins were eluted by boiling in 1x Laemmli buffer at 95 °C for 30 min.
8
Accelerated Native Protein Isolation
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Accelerated native isolation of proteins on nascent DNA (aniPOND) has been carried out as described by Leung KH et al.49 (link) Briefly, wild-type and SV40, H-RASV12-transformed MEF (around 3 × 107 cells for each experimental condition) were pulsed with 10 μM of EdU (Life technologies, A10044) for 20 or 10 min, respectively. Click-IT reaction was performed using biotin azide (Life Technologies, B10184) or dimethylsulphoxide as a negative control. Chromatin was sonicated following the procedure described by Leung KH et al.49 (link) Biotinylated Edu was captured overnight at 4 °C using streptavidin-coated magnetic beads (Life Technologies, 65602). Chromatin proteins bound to EdU were eluted and analysed by immunoblot.
9
EdU Incorporation and Click-iT™ Labeling
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5-ethynyl-2'-deoxyuridine (EdU, Life Technologies, #A10044) was added to cells at a final concentration of 20μM for 10 min prior to harvest. After fixation, cells were subjected to the Click-iT™ reaction using Biotin-Azide (Life Technologies, #B10184) for 1 h at room temp, according to the manufacturer's instructions. Cells were then stained with the appropriate antibodies using normal immunocytochemistry techniques.
10
Palmitic Acid Analogue Labeling and Enrichment
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Transiently transfected HEK293E cells were treated with 25 µm of the palmitic acid analogue, 17‐ODYA (Cayman Chemical) or mock treated with an equal volume of DMSO, 24 h after transfection. The 17‐ODYA labelling was allowed to continue for 6 h at 37°C under standard culture conditions. Cell pellets were collected and lysed in 1% Triton X‐100/50 mm Tris‐Cl pH 7.4/150 mm NaCl/protease inhibitor cocktail (Roche) for 30 min at 37°C with shaking. The click chemistry reaction was then performed as previously described (Martin and Cravatt, 2009 ). Briefly, 2 mg of each protein sample was incubated with the following chemicals in PBS: 100 µm biotin‐azide (Life Technologies), 1 mm Tris(2‐carboxyethyl)phosphine (TCEP; Sigma‐Aldrich), 100 µm Tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA, Sigma‐Aldrich) dissolved in DMSO/tert‐butanol (20%/80%), and 1 mm CuSO4 (Sigma‐Aldrich). The reaction was allowed to continue for 1.5 h at room temperature with shaking. Biotinylated proteins were affinity purified by incubating with a 50 µl bed volume of streptavidin‐agarose (Thermo Scientific) for 2 h at room temperature with rotation. Proteins were eluted by incubating with 100 µl of 2% SDS/50 mm Tris‐Cl pH 7.4/5 mm EDTA for 5 min at 95°C with shaking.
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