Cell supernatants were mixed with a rabbit polyclonal anti-CEP antibody and incubated at 37 °C for 1 hour with gentle shaking. ELISA plates were coated with 221 nM CEP- BSA and blocked with 2% BSA. After 1 hour, the plates were washed three times, filled with the supernatant/antibody mixtures, and incubated at room temperature (RT) for 1 hour. Then, the plates were washed three times, filled with a HRP-conjugated anti-rabbit secondary antibody (Invitrogen), and incubated at RT for 1 hour. For ELISA standard, we used serial dilutions of CEP-BSA starting from 1–5 μM as maximum and plotted a standard curve. CEP concentrations were measured by reading absorbance at 450 nm by using spectroscopy after the plates were washed and incubated with HRP substrates (ABTS, Invitrogen). The same procedure was performed for EP measurements.
Hrp conjugated anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HRP-conjugated anti-rabbit secondary antibody is a reagent used in various immunoassay techniques, such as Western blotting and ELISA, to detect the presence of rabbit primary antibodies. The antibody is conjugated with horseradish peroxidase (HRP), an enzyme that can catalyze a colorimetric or chemiluminescent reaction, allowing for the visualization and quantification of the target proteins.
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Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Mini trans blot cell, by Bio-Rad (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Ibright 1500 system, by Thermo Fisher Scientific (2 mentions)
Np 40 buffer, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Centrifuge 5424r, by Eppendorf (2 mentions)
Pierce ecl western blotting substrate, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Thermo Fisher Scientific (1 mentions)
Ab5103, by Abcam (1 mentions)
Af 401 na, by Cell Signaling Technology (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
29 protocols using hrp conjugated anti rabbit secondary antibody
1
Quantifying Carboxylethylpyrrole (CEP) Levels
2
Western Blot Analysis of Inflammasome Activation
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Murine cell and tissue lysates were prepared with radioimmunoprecipitation assay (RIPA) buffer. Protein lysates were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were probed with anti-CitH3 (Abcam #ab5103, 1:1000 dilution), anti-Caspase-1 (Abcam # ab179515, 1:1000 dilution), anti-IL-1 β (R&D system, # AF-401-NA, 1:1000 dilution) and anti- β -actin (Cell Signaling Technology, #3700, 1:1000 dilution) antibodies, followed by HRP-conjugated anti-rabbit secondary antibody (Invitrogen, #G-21234) or Dylight 800-conjugated anti-mouse secondary antibody (Cell Signaling Technology, #5257S). Images were visualized with ChemiDoc™ Touch Imaging System (Bio-Rad) and analyzed with Image Lab (Bio-Rad).
3
Histone Acetylation Regulation by TSA
4
Immunoblotting of tagged proteins
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The purified complexes and protein extracts were analyzed by 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on a nitrocellulose membrane. Proteins were detected using standard immunoblotting techniques. GST-tagged proteins were detected with a rabbit polyclonal anti-GST antibody (Sigma-Aldrich) and a HRP-conjugated anti-rabbit secondary antibody (Invitrogen). 3xFlag-tagged proteins were detected with a mouse monoclonal HRP-conjugated anti-3xFlag antibody (Sigma-Aldrich).
5
Western Blot Analysis of IRF5 in DCs
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Fresh isolated or treated DCs were rinsed with cold PBS and lysed in lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration was measured using NanoDrop. Equal amounts of protein were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes; nonspecific sites were blocked with 5% BSA in TBST and the membranes were then incubated with dilutions of the primary antibody as recommended by the manufacturer. The antibodies used are as follows: anti-IRF5(1:1000, 96527), anti-β actin (1:5000, 3700) (CellSignaling, Beverly, MA, USA), HRP conjugated anti-rabbit secondary antibody (1:2000, Invitrogen, Grand Island, NY, USA). Western blots were visualized using the enhanced chemiluminescent (ECL) reagent (Thermo Fisher).
6
Histone Deacetylase Inhibitor TSA Regulates TP53 Acetylation
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Human HEK293 and HCT116 cells were cultured in EMEM (ATCC, 30‐2003) and DMEM Gibco # 11995‐065) medium, respectively. Cells were treated with 4 μM histone deacetylase inhibitor Trichostatin A (TSA) (Sigma, T8552) or DMSO for 24 hours, then collected and lysed in NP40 buffer (Fisher, FNN0021) for Western blot analysis. Zebrafish wild‐type 24 hpf embryos were treated with 0.5 μM TSA for 24 and 48 hours. A 20 μg of total protein extracted from cell and zebrafish embryo samples was run on a 10% SDS‐PAGE gel, blotted to PVDF membrane (Bio Rad, 1620176), and incubated with rabbit monoclonal anti‐TP53‐acetylated‐K370 (abcam, ab183544) at 1:500, rabbit polyclonal anti‐β actin (Cell Signaling, 4967) at 1:2000, rabbit polyclonal anti‐RBBP4 (Bethyl A301‐206A‐T, RRID: AB_890631) 1:2000, and HRP‐conjugated anti‐rabbit secondary antibody (Invitrogen, 31 460) at 1:5000. Blots were developed with SuperSignal West Dura Extended Duration Substrate (ThermoScientific, 34075) and imaged on a ThermoFisher iBright 1500 system.
7
Binding Assay for IgSF8 Interactions
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For direct binding assays of IgSF8 to Tenascin-R and Brevican, 1 µg recombinant His-tagged mouse IgSF8 (Sino Biological) was incubated in 1 ml binding buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, and 0.1% Tween-20) with equimolar amounts of control Fc-protein (Jackson ImmunoResearch), purified Tenascin-R-Fc or Brevican-Fc, and rotated end-over-end for 1 h at RT. Protein-A/G agarose beads (100 µl slurry; Santa Cruz Biotechnology) were added and rotated end-over-end for 1 h at 4 °C. Beads were washed 4× in binding buffer and 1× in PBS, and boiled in 50 µl 2× sample buffer. Samples were analyzed by WB. Rabbit anti-6x-His Tag primary antibody (1:250; Thermo-Fisher) and HRP-conjugated anti-rabbit secondary antibody (1:10,000; Thermo-Fisher) were used to detect IgSF8-His. Uncropped and unprocessed scans are provided in Source data file.
8
Quantifying Egr1 Protein Levels
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Whole-cell lysates were obtained by resuspending cell pellets in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche, Basel, Switzerland) as previously described.29 Typically, 100 μl RIPA buffer was used for 1 × 106 cells. Moreover, 30 μg of protein was loaded in each lane and separated by 8% SDS-PAGE gel with all-blue protein markers (Bio-Rad, Hercules, CA, USA). Proteins were transferred to nitrocellulose membranes (Bio-Rad) in a Mini-Trans-Blot Cell (Bio-Rad). The membranes were blocked with 5% fat-free milk powder in TBS at room temperature for half an hour and then incubated with the following primary antibodies at 4 °C overnight: anti-Egr1 (Proteintech, Wuhan, China, 55117-1, 1:500) and anti-β-actin (Sigma, Burlington, MA, USA, A1978, 1:5,000). The next day, the membranes were washed with TBS and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Thermo Fisher, 61-6520, 1:5,000) or anti-mouse secondary antibody (Thermo Fisher, 31464, 1:5,000) for 1 h at room temperature. For densitometrical quantification, densities of target proteins were normalised to those of β-actin. Data are expressed as relative protein levels compared with the control group, which is arbitrarily set as 1.
9
Immunostaining of RH30 and RD Cells
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Cytocentrifuge preparations of RH30 and RD cells were fixed on slides and permeabilized with acetone for 10 minutes and then incubated with anti-FGF8 antibody (Peprotech) diluted in PBS1X for 1 h at room temperature. After washing, slides were probed at room temperature for 40 minutes with HRP-conjugated anti-rabbit secondary antibody (Thermo Fisher scientific) and then with 3,3’-diaminobenzidine (DAB) (Dako) A brief dive in Gill’s hematoxylin solution (Sigma-Aldrich) was done for visualization of nuclei in cell preparations. The images were acquired with a Leica DFC420 digital camera, mounted on a Leica DMLB microscope, at 20X magnification. Image analysis was performed with Leica IM1000 software (Leica Microsystem Ltd).
10
Protein Expression Analysis in Cell Lines
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