To assess the facilitation of TrxA proteins in Salmonella, the abundance of FLAG-OmpR was detected by immunoblot assay with the indicated strains. Cells grown in LB broth overnight at 37 °C were disrupted by sonication in PBS, and half of lysates were centrifuged for 10 min at 13,000 g to collect cell-free extracts. Protein concentrations of crude extracts and cell-free extracts were determined with BCA protein assay kit (cat. # 23227, Pierce, Rockford, IL) then the specimens were loaded onto 12% SDS-PAGE gels. The blots were treated with a 1:500 dilution of anti-FLAG monoclonal antibody (cat. # F1804, Sigma, St. Louis, MO), followed by a 1:20,000 dilution of goat anti-mouse IgG conjugated with horseradish peroxidase (cat. # 45-000-692, Fisher Scientific, Grand Island, NY). As an internal control, the blots were treated 1: 5000 with an anti-DnaK monoclonal antibody (cat. # ab69617, Abcam, Waltham, MA), followed by a 1:20,000 dilution of goat anti-mouse IgG conjugated with horseradish peroxidase. The specimens were visualized using the ECL prime Western blotting detection reagent (cat. # RPN2232, GE Healthcare Life Sciences, Marlborough, MA) in the ChemiDoc XRS imaging system (Bio-Rad, Hercules, CA). Protein density was measured by the ImageJ program (NIH).
Horseradish peroxidase
Manufactured by Abcam
Sourced in United States, United Kingdom
Horseradish peroxidase (HRP) is a widely used enzyme in various biochemical and molecular biology applications. It is an oxidoreductase enzyme that catalyzes the oxidation of substrates in the presence of hydrogen peroxide. HRP can be used as a reporter enzyme in immunoassays, western blotting, ELISA, and other detection methods due to its ability to produce a colorimetric, chemiluminescent, or fluorescent signal.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (6 mentions)
Gapdh, by Abcam (5 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Hrp conjugated goat anti rabbit igg, by Abcam (4 mentions)
Ab8226, by Abcam (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Anti glut1, by Abcam (3 mentions)
Anti bcl 2, by Abcam (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Rapidstep ecl reagent, by Merck Group (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Bcl2 associated x (bax), by Abcam (2 mentions)
Anti il 1β, by Abcam (2 mentions)
H k atpase α, by Abcam (2 mentions)
H k atpase β, by Abcam (2 mentions)
Bcl 2, by Abcam (2 mentions)
75 protocols using horseradish peroxidase
1
Immunoblot Analysis of TrxA-Mediated OmpR Abundance in Salmonella
2
Western Blot Analysis of Protein Expression
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Western blot analysis was carried out to assess protein expression as previously described [16 (link)]. Sodium dodecyl sulfate polyacrylamide was applied for isolation of proteins followed by transferring to the poly vinylidene difluoride membrane. After being sealed by milk, proteins were grown with primary antibodies overnight at 4°C. Post washing, secondary antibodies conjugated with horse radish peroxidase (Abcam, Shanghai, China) were supplemented for 1 h followed by exposure to an enhanced chemiluminescence detection system. The primary antibodies included anti-Ube2b (1/1000, ab128951, Abcam), anti-DNMT3a (1/2000, ab188470, Abcam), anti-Kcna2 (1 µg/ml, ab192758, Abcam), and anti-β-actin (1 µg/ml, ab8226, Abcam).
3
Immunoblotting Analysis of IFNλ4 Signaling
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The cells were lysed with RIPA buffer (Thermo Fisher Scientific) to prepare total cell lysates. Ten micrograms of each cell lysate were loaded on SDS-PAGE gels prior to immunoblotting. The antibodies used for immunoblotting were: IFNλ4 (1:200, mouse, Millipore MABF227), IFNλ4 (1:200, rabbit, Abcam ab196984), STAT1 (1:1000, rabbit, BD Biosciences 610120), PY-STAT1 (1:1000, mouse, BD Biosciences 612233), STAT2 (1:1000, rabbit, Santa Cruz Biotechnology sc-476), IRF9 (1:1000, rabbit, Santa Cruz sc-496), SOCS1 (Abcam #62584), USP18 (Cell Signaling Technology #4813), horseradish peroxidase (HRP)-conjugated rabbit IgG (1:5000, Abcam ab97051), and HRP-conjugated mouse IgG (1:5000, Abcam ab97023).
4
Developmental Expression of Mitochondrial Dynamics Proteins
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Brains from chicken embryos obtained on days 8, 12, and 18 of incubation were dissected in cold PBS (Gibco). Brains were homogenized in RIPA lysis buffer on ice for 30 min with a protease inhibitor cocktail (Thermo Fisher™), and centrifuged at 12,000 g for 0.5 h. Samples were boiled and denatured in loading buffer containing 5% 2-mercaptoethanol (Invitrogen) and subjected to electrophoresis through a 10% SDS polyacrylamide gel. The separated proteins were transferred to a PVDF membrane (Bio-Rad) and probed with primary antibodies for MFN1 (1/600, Proteintech), MFN2(1/600, Proteintech), OPA1 (1/600, Proteintech), DRP1 (1/800, Proteintech), α-Tubulin (1/6,000, Abcam) and the appropriate secondary antibody conjugated with horseradish peroxidase (1/5,000, Abcam). Bound proteins were detected using a Pierce ECL Western Blotting Substrate kit (Thermo Scientific™). The results were visualized by scanning and then analyzed using Image J software (NIH).
5
Western Blot Analysis of Glucose Metabolism Proteins
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Cells and tumor tissues were lysed using RIPA buffer (Beyotime). Lysates were mixed with loading buffer (Thermo Fisher), which was then boiled in boiling water. Then samples were loaded on 12% bis–tris-acrylamide gel (Thermo Fisher) and then electrotransferred onto polyvinylidene fluoride (Millipore, Bradford, MA, USA). After immersed in 5% nonfat milk diluted with tris buffered saline tween (Millipore), membranes were incubated with anti-recombinant glucose transporter 1 (anti-GLUT1) (1:200; Abcam), anti-hexokinase 2 (anti-HK2) (1:1000; Abcam), anti-YWHAZ (1:1000; Abcam) and anti-β-actin (1:1000; Abcam). Secondary antibody labeled with horseradish peroxidase (1:10,000; Abcam) was subsequently used to incubate membranes. Protein bands were visualized using RapidStep ECL Reagent (Millipore). β-actin acted as a control.
6
Western Blot Analysis of NSCLC Cell Proteins
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NSCLC cells were lysed with RIPA buffer (Beyotime). The protein sample was boiled and loaded by 12% sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein was transferred into nitrocellulose membranes (Membrane Solutions, Shanghai, China). Bands were blocked in 5% skim milk. Then, bands were incubated with anti-c-myc (1:1000; Abcam, Cambridge, UK), anti-E-cadherin (1:1000; Abcam), anti-Vimentin (1:5000; Abcam), anti-caspase-3 (anti-casp-3) (1:10000; Abcam), anti-multi-drug resistance gene-1 (anti-MDR1; 1:500, Abcam), anti-HIF-1α (1:10000; Abcam) and anti-GAPDH (1:10000; Abcam). Following proteins were reacted with secondary antibody labeled with horseradish peroxidase (1:1000; Abcam). Proteins were visualized with enhanced chemiluminescence (KeyGen, Nanjing, China). GAPDH was used as a reference.
7
Immunohistochemistry for Osteopontin
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For antigen retrieval, the specimens were deparaffinized and treated with citrate buffer 0.01 mol/l, pH 6.0 (Merck), heated at 100°C for 6 × 2 min.
For blocking of endogenous peroxidase, sections were incubated in H2O2 3% for 10 min. The mouse monoclonal antibody against osteopontin (1:300 concentration; Santa Cruz Biotechnology, USA) was used at 4°C overnight. Then, specimens were incubated in secondary antibody conjugated with horse radish peroxidase (Abcam) for 30 min. After staining with diaminobenzidine (Sigma), hematoxylin dye was used to counter staining.
For blocking of endogenous peroxidase, sections were incubated in H2O2 3% for 10 min. The mouse monoclonal antibody against osteopontin (1:300 concentration; Santa Cruz Biotechnology, USA) was used at 4°C overnight. Then, specimens were incubated in secondary antibody conjugated with horse radish peroxidase (Abcam) for 30 min. After staining with diaminobenzidine (Sigma), hematoxylin dye was used to counter staining.
8
Immunoblotting of Viral Proteins in Plants
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Total protein was extracted from infiltrated leaf patches or TuMV-infiltrated or systemically infected N. benthamiana leaves as described previously [60 (link)]. Immunoblotting was performed with rabbit GFP polyclonal antibodies (ab6556, Abcam) or Myc polyclonal antibodies (ab9106, Abcam), followed by goat anti-rabbit (ab6721) secondary antibody conjugated to horseradish peroxidase (Abcam). Blotted membranes were washed thoroughly and visualized by chemiluminescence.
9
Western Blot Analysis of Apoptosis Regulators
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The total cell lysate was prepared using RIPA buffer as described [49 (link)] and Western blotting was carried out as detailed [50 (link)]. The following antibodies were used: rabbit polyclonal anti-Beclin1 (1:2000, Abcam), Anti-Bcl2 (1:500, Abcam), Anti-CD9 antibody (1:250 Abcam) and anti-rabbit or anti-mouse secondary antibodies conjugated to horse-radish-peroxidase (1:20,000, Abcam) and β-Actin (1:5000, Santacruz Inc.,). Following TBST washes, protein bands were visualized using ECL and ChemiDoc Imaging System and quantified with Image-J.
10
Western Blot Analysis of rSm16
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0-3hRP fractions and rSm16 were transferred after SDS-PAGE onto nitrocellulose membranes using an iBlot® Transfer Stack (Life Technologies). The membranes were then processed using the SnapID® system (Millipore) blocked with PBS containing 1% BSA, incubated first with rabbit anti-rSm16 antibody (1:5000) (gift from Dr Martin Gullberg, Umeå University, Sweden) for 10 min, and then goat anti rabbit antibody (1:30000) conjugated to horseradish peroxidase (Abcam). SuperSignal® West Pico chemiluminescence reagent (Thermo Scientific) was used to reveal labelled proteins using X-ray film imaging (GE Healthcare).
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