Approximately 2 × 1011 pfu of PD‐1 or helper phage particles were boiled for 10 min in sodium dodecyl sulfate (SDS) loading buffer and were clarified by centrifugation. After separation in a 12% SDS–PAGE, the gel was transferred to a PVDF membrane at 200 mA for 2 hours. The membrane was incubated with blocking buffer for 1 hour at room temperature, followed by incubation overnight with anti‐pIII Ab (NEB, Ipswich, MA, USA) at 1:1000 dilution in blocking buffer. After 3 washes, the membrane was incubated for 1 hour in blocking buffer containing HRP‐conjugated secondary antibody (Multi Sciences, Hangzhou, China) at 1:800 dilution. Color was developed with an ECL detection kit (Multi Sciences).
Hrp conjugated secondary antibody
Manufactured by MultiSciences Biotech
Sourced in China
The HRP-conjugated secondary antibody is a laboratory reagent used for signal amplification in various immunoassay techniques. It consists of a secondary antibody that is conjugated with the enzyme horseradish peroxidase (HRP). The HRP enzyme can catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and quantification of target analytes in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
β actin, by MultiSciences Biotech (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Chemiluminescence kit, by PerkinElmer (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ab8245, by Abcam (2 mentions)
Ecl detection kit, by MultiSciences Biotech (1 mentions)
Ecl reagent kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
Ripa lysis buffer, by Takara Bio (1 mentions)
Lc3ii lc3i, by Abcam (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Stat3, by Abcam (1 mentions)
Akr1c1, by Abcam (1 mentions)
P jak2, by Abcam (1 mentions)
P stat3, by Abcam (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Quantity one bioanalysis software, by Bio-Rad (1 mentions)
Calponin, by Abcam (1 mentions)
12 protocols using hrp conjugated secondary antibody
1
Detecting Phage Particle Proteins
2
Western Blot Analysis of Protein Expressions
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Huh7 and SMMC7721 cells were lysed in a RIPA lysis buffer (Takara Bio, Japan) for the extraction of total protein. The protein concentration was quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, CA, USA). Total proteins were separated by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were incubated with blocking buffer (5% nonfat dry milk dissolved in 1× Tris buffered saline with 0.1% Tween-20) at room temperature for 1 h, followed by the primary antibodies, including LC3II/LC3I, P62, AKR1C1, p-JAK2, p-STAT3, JAK2, STAT3, and GAPDH (1 : 1000, Abcam, UK), at 4°C overnight. Then, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1 : 500, MultiSciences, China) for 1 h at room temperature in the dark. Protein bands were presented using ECL reagent kit (Thermo Fisher Scientific, CA, USA), and images were captured using a ChemiDoc™ imaging system (Bio-Rad, CA, USA). The relative protein expression was quantified by calculating the band density normalized to GAPDH.
3
Western Blot Analysis of SSADH Protein
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Cells were lysed with RIPA lysis buffer (Beyotime, Beijing, China) containing 1% PMSF (Beyotime, Beijing, China). Protein (15 μg) was loaded on 10% SDS-PAGE gels for electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk in TBS containing 0.1% Tween 20 for 1.5 h at room temperature and then incubated with antibody for SSADH (AffinitY, Cat# DF12820) and β-actin (Multi Sciences, Hangzhou, China) at 4 °C overnight. The membranes were then incubated with HRP-conjugated secondary antibody (Multi Sciences, Hangzhou, China) for 2 h at room temperature. Signals were detected using film by darkroom exposure (Servicebio, G2019, China). In each experiment, each sample run on the SDS-PAGE gel in duplicate.
4
Arctiin Modulates H9N2 Virus in A549 Cells
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Confluent monolayers of A549 cells in a six-well plate, infected with H9N2 viruses, were treated with various concentrations of arctiin. After 24 h, the cells were washed twice with PBS and lysed in ice-cold RIPA buffer (Beyotime, shanghai, China) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich). Cellular lysates were collected and centrifuged for 15 min at 10,000 g at 4 °C to remove debris. Then, protein concentration of the supernatant was measured by BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Afterward, 20 μg of total protein from each sample was fractionated by 10% SDS-PAGE. After transfer of the resolved protein onto 0.2-μm PVDF membranes, the membranes were blocked with 5% nonfat milk and incubated with primary antibodies overnight at 4 °C. Then, the membranes were washed three times with 1 × TBST (Tris-buffered saline; 20 mM Tris-HCl PH7.4, 150 mM NaCl, 0.1% Tween 20) and further incubated with HRP-conjugated secondary antibody (Multiscience, Hangzhou, China) (1:5000 dilution) for 1 h at room temperature. The bands were detected by a chemiluminescence kit (PerkinElmer).
5
Western Blot Analysis of VSMC Markers
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VSMCs lysis was conducted using RIPA lysis buffer (Beyotime, Shanghai, China) supplied with 1% protease inhibitors. The proteins were separated by electrophoresis and transferred onto PVDF membranes by electroblotting for 3 h at 150 mA. Then, the membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% skim milk. The membranes were incubated with primary antibodies against BMP7 (Abcam, Cambridge, MA), SM22α (Abcam), calponin (Abcam), OPN (Cell Signaling Technology, Danvers, MA, USA), Runx2 (Cell Signaling Technology), and GAPDH (ZSGB-Bio, Beijing, China), and then the HRP-conjugated secondary antibody (MultiSciences, Beijing, China). Chemiluminescent signals were detected and quantified by densitometry using Quantity One Bioanalysis software (Bio-Rad).
6
Arctiin Inhibits H9N2 Virus in A549 Cells
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Con uent monolayers of A549 cells in a six-well plate, infected with H9N2 viruses, were treated with various concentrations of arctiin. After 24 h, the cells were washed twice with PBS and lysed in ice-cold RIPA buffer (Beyotime, shanghai, China) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and 1 mM phenylmethanesulfonyl uoride (PMSF; Sigma-Aldrich). Cellular lysates were collected and centrifuged for 15 min at 10,000g at 4°C to remove debris. Then, protein concentration of the supernatant was measured by BCA Protein Assay Kit (Thermo Scienti c, Waltham, MA, USA). Namely, 20 µg of total protein from each sample was fractionated by 10% SDS-PAGE. After transfer of the resolved protein onto 0.2-µm PVDF membranes, the membranes were blocked with 5% nonfat milk and incubated with primary antibodies overnight at 4°C. Then, the membranes were washed three times with 1 ⋅ TBST and further incubated with HRP-conjugated secondary antibody (Multiscience, Hangzhou, China) (1:5000 dilution) for 1 h at room temperature. The bands were detected by a chemiluminescence kit (PerkinElmer).
7
IPEC-J2 Cell Culture and Transfection
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IPEC-J2 cell line (Guangzhou Jennio Biotech Co, Ltd., China) used in this study was cultured in Dulbecco’s Modified Eagle’s Medium nutrient (DMEM from Life Technologies, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 16 mM HEPES (Life Technologies), and 100 μg/ml penicillin-streptomycin (Life Technologies), and incubated in an atmosphere of 5% CO2 at 37 °C. Cells were routinely seeded at a density of 2 × 105/mL in plastic tissue culture flasks (25 cm2 flasks, Corning, Shanghai, China) and passaged every 3–4 days for a maximum of 25 passages. In our experiments, IPEC-J2 cells were grown on 24-,6- well or 60 mm plastic tissue culture plates (Corning) at a density of 3 × 105/well, 1.5 × 106/well or 7.5 × 106/well, respectively. Poly(I:C) (HMW)/LyoVec (InvivoGen). Anti-TGEV nucleocapsid protein (N) antibodies (Preservation of our laboratory), anti-actin antibodies (Multisciences, Hangzhou, China) and HRP-conjugated secondary antibodies (Multisciences). pISRE-TA-Luc (Beyotime Technologies). pIFNβ-Luc was conducted by traditional method inserting a 348 bp promoter sequence of IFN-β into pGL6-TA-Luc plasmid. plentiCRISPR V2 used for IFNAR1/2 knockout was conducted following the method described in Feng Zhang Lab.
8
Protein Extraction and Western Blot Analysis
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Total protein from A549 cells was extracted on ice by the addition of lysis buffer (Beyotime, China) containing protease inhibitors (Sigma, USA). Twenty micrograms of protein was heat denatured in sodium dodecyl sulfate loading buffer (Solarbio, Beijing, China), separated by 10% SDS–polyacrylamide gel electrophoresis, and transferred to PVDF membranes (0.2 μm, Bio-Rad, USA). Membranes were incubated overnight with the indicated primary antibodies, followed by incubation with HRP-conjugated secondary antibodies (Multi-science, Hangzhou, China). Protein bands were visualized by an ECL kit (Perkin Elmer Life Sciences). The original images for western blot are shown in Additional file 1 .
9
Protein Expression Analysis by Western Blot
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Total proteins of each cultured cell were extracted with RIPA Lysis Buffer (Applygen Technologies Inc., Beijing, China). After the protein concentration was determined by BCA kit (MultiSciences, Hangzhou, China), the target proteins were classified by 15% or 8% SDS-PAGE (MultiSciences) and then transferred to PVDF membranes (Millipore, Darmstadt, Germany) and blocked with 5% skimmed milk powder at 25°C for 90 minutes. The membranes were incubated overnight at 4°C with primary antibodies, anti-E-Cadherin (ab40772, 1 : 20000), anti-N-Cadherin (ab76011, 1 : 10000), anti-HMGA1 (ab168260, 1 : 1000), and anti-GAPDH (ab8245, 1 : 5000) (Abcam, Cambridge, UK). The membranes were then incubated with HRP-conjugated secondary antibodies (1 : 1000, MultiSciences) at 25°C for 90 min, and the bands were visualized by a chemiluminescence.
10
Quantification of DRG Protein Levels
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Rats were sacrificed by an overdose of chloral hydrate. Lumbar DRGs (L4-L6) from the ipsilateral side were quickly dissected out and frozen in liquid nitrogen. Each sample was lysed and centrifuged at 12,000 r/min for 30 min at 4℃ to collect the supernatant; 20 mg of protein was fractionated on 10% polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and then transferred to polyvinyl difluoride (PVDF) membranes (Bio-Rad, USA) for 2 h at 200 mA. The membranes were blocked in TBS (50 mM Tris-Base, 133 mM NaCl, pH = 7.4) containing a 5% dilution of non-fat milk powder for 2 h at room temperature (RT) and then incubated with primary antibody (anti-GRK6 at 1:500, Santa Cruz Biotechnology, USA; CXCR1 at 1:500, Santa Cruz Biotechnology, USA; anti-CXCR2 at 1:100, Boster, P.R. China; anti-CXCR4, Santa Cruz Biotechnology, USA; anti-GAPDH at 1:1000, Goodhere, China; anti-β-actin at 1:1000, Multi-Sciences, P.R. China) in TBS containing 1% milk at 4℃ overnight. After washing in TBST (0.5% Tween-20 in TBS), the PVDF membranes were incubated with HRP-conjugated secondary antibodies (1:4000, Multi Sciences Biotech Co., P.R. China) in TBS and 1% milk for 2 h at RT. Bands were visualized using ECL (Biological Industries, P.R. China) and exposed to Kodak X-ray films. Films were scanned and band intensities of target proteins were analyzed using Optic Quant software (ImageJ, NIH, USA).
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