Frozen human muscle samples and whole Drosophila were homogenized in RIPA buffer (25 mMTris‐HCl pH 7.6, 150 mMNaCl, 1% NP‐40, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 50 mMNaF, 1 mM Na3VO4). After 20 min of centrifugation at 14,000 g at 4ºC, the supernatant was collected and protein concentration was determined with the BioRad DC Protein Assay kit. Tissue lysate was diluted with the SDS‐PAGE sample buffer (125 mM Tris‐HCl pH 6.8, 5% 2‐mercaptoethanol, 2% SDS, 10% glycerol, 0.01% bromophenol blue) and heated at 95ºC for 10 min. Equal amounts of total proteins were loaded and resolved on a 4%–12% SDS‐polyacrylamide gel (Life Technologies), then transferred onto a nitrocellulose membrane (20 μm). Primary antibodies used for immunoblotting were as follows: anti‐SERCA1 (1:800, a polyclonal antibody targeting residues surrounding Leu24 near the N terminus, Cell Signaling), anti‐SERCA1 (1:20, Developmental Studies Hybridoma Bank), anti‐GAPDH (1:1,000, Cell Signaling), anti‐β‐actin (1:200, Santa Cruz). For detection, either anti‐mouse peroxidase‐conjugated secondary antibodies or anti‐rabbit peroxidase‐conjugated secondary antibodies (Sigma‐Aldrich) in combination with ECL (Thermo Scientific) were used. Western blot assays were repeated in triplicate.
Anti rabbit peroxidase conjugated secondary antibody
Manufactured by Merck Group
Sourced in United States
The Anti-rabbit peroxidase-conjugated secondary antibody is a laboratory reagent used to detect and quantify target proteins in various immunoassays. It contains a peroxidase enzyme conjugated to an antibody that specifically binds to rabbit primary antibodies. This reagent can be used to amplify and visualize the signal from rabbit primary antibodies in techniques such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Aec substrate, by Abcam (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Bx43 upright microscope, by Olympus (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Sds polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Anti serca1, by Developmental Studies Hybridoma Bank (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Gs 800 densitometer, by Bio-Rad (1 mentions)
Polyscreen membrane, by PerkinElmer (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Mini trans blot system, by Bio-Rad (1 mentions)
Rabbit anti cleaved caspase 3 polyclonal antibody, by Cell Signaling Technology (1 mentions)
Etoposide, by Merck Group (1 mentions)
Precision plus protein standard, by Bio-Rad (1 mentions)
Whole cell lysis buffer, by Beyotime (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
17 protocols using anti rabbit peroxidase conjugated secondary antibody
1
Quantitative Western Blot Analysis of SERCA1
2
Quantitative Western Blot Analysis of Gut and Liver Transporters
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A total of 15 µg of total proteins extracted from the ileal mucosa, the liver, or the kidney were prepared in ice-cold buffer (0.154 M KCl, 0.01 M phosphate buffer, pH = 7.4), separated on 10% SDS-PAGE, then blotted onto a Polyscreen membrane (PerkinElmer Life Sciences). Rabbit polyclonal anti-ASBT, NTCP, BSEP, MRP2, OSTα, and OSTβ were a generous gift from Dr. M. Souidi (Institut de Radioprotection, Fontenay-aux-Roses, France), Prof. M. Trauner (Gastroenterology and Hepatology, Vienna, Austria), Prof. N. Ballatori University of Rochester School of Medicine, Rochester, NY, USA), and Prof. G. Kullak-Ublick (University Hospital Zurich, Zurich, Switzerland). Anti-b-actin antibodies and anti-rabbit peroxidase-conjugated secondary antibodies were purchased from Sigma-Aldrich. ECL blotting kit (PerkinElmer Life Sciences) was used for detection. Blot quantification was performed using calibrated GS-800 densitometer (Biorad, Marnes-la-coquette, France).
3
Cleaved Caspase-3 Immunoblotting in AVICs
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AVIC cultures were subjected to cell lysis and protein quantification as described above. Supernatants were then separated by SDS-PAGE and transferred on nitrocellulose membranes by electroblotting using the Bio-Rad Mini Trans-Blot system (Hercules, CA). After blocking with 3% skim milk (Sigma) solution, blotted membranes were incubated with 1:500 rabbit anti-cleaved-caspase-3 polyclonal antibody (Cell Signaling Technology, Danvers, MA) overnight at 4C and then with 1:15,000 anti-rabbit peroxidase-conjugated secondary antibody (Sigma) for 1 hr at room temperature. Immunoreactive bands were revealed using the Pierce enhanced chemiluminescence assay (Thermo Fisher Scientific) with a maximum exposure time of 4 hr. For accurate estimation of protein molecular weight, Precision Plus Protein Standards (Bio-Rad) were used. As positive controls, lysates of AVICs treated with 50-µM etoposide (Sigma) for 18 hr were used.
4
Protein Quantification and Immunoblotting
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After washing in PBS solution, total protein of cells was extracted using whole cell lysis buffer (Beyotime). Bio-Rad protein assay was applied to quantify the protein concentration. Immunoblotting was done as previously described [26 (link)]. The following antibodies were used: anti-HIF-1α, abti-NRF2 antibodies (1:500 polyclonal; Bethyl), anti-VEGF and anti-PDGF antibodies (1:1000 monoclonal; Abcam), and anti-rabbit peroxidase-conjugated secondary antibody (1:10,000; Sigma).
5
Quantifying HDAC11 Expression in HCC Cells
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Total proteins were extracted from HCC cells using RIPA lysis buffer (Beyotime, China). Western blot was used to analyze HDAC11 expression as previously described (Lou et al., 2019b (link)). The primary antibodies of HDAC11 (1:1,000) and GAPDH (1:2,000) were purchased from Abcam, and anti-rabbit peroxidase-conjugated secondary antibody was purchased from Sigma (1:5,000). The band density of HDAC11 was normalized to GAPDH and then quantified using ImageJ software.
6
GPX3 Expression in Breast Cancer Cells
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Protein of breast cancer cells was extracted using RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Thermo Scientific, USA). Western blot was performed as previously described [18 (link)]. The primary antibodies of GPX3 (1:1000) and GAPDH (1:1000) were purchased from Abcam, and anti-rabbit peroxidase conjugated secondary antibody was purchased from Sigma (1:5000). GPX3 band density was normalized to GAPDH and quantified by ImageJ software.
7
Western Blot Analysis of Cell Signaling
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Cells were washed in PBS solution, after which proteins were extracted using an established protocol. The proteins were then mixed with Laemmli sample buffer, heated at 65°C for 10 min, loaded (20 μg for each sample), separated by sodium dodecyl sulfate-polyacrylamide gel (7.5%) electrophoresis under denaturing conditions, and electroblotted onto nitrocellulose membranes. The membranes were blocked by incubation in blocking buffer (1% BSA in Tris-buffered saline-0.1% Tween 20), then incubated with primary antibodies, washed, and incubated with anti-rabbit peroxidase-conjugated secondary antibody (1:10,000; Sigma). Immunoblots were developed using a BeyoECL (Beyotime) and Tanon 5200 system. The primary antibodies were as followed: α-SMA (Novus, 1:500), Col1a1 (Abcam, 1:1000), TGF-β (Abcam, 1:500), Cyclin D1 (Abcam, 1:200) p-IκB (Santa crus, 1:200), MyD88 (Abcam, 1:500), TLR4 (Abcam, 1:300).
8
Quantifying PPAR-gamma Expression in MCAO
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The regions of ischemia and penumbra in the right side of each MCAO brain (n=8 per group) were isolated on ice and homogenized in lysis buffer. Protein concentrations were measured with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equivalent amounts of protein were separated on 10% SDS-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were probed with rabbit anti-PPARγ (1:1000 Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight and subsequently incubated with anti-rabbit peroxidase-conjugated secondary antibody (1:2000, Sigma-Aldrich) for 1 h at room temperature. The blots were quantified and normalized with glyceraldehyde-3-phosphate dehydrogenase (GADPH) by scanning with a ScanJet scanner (Hewlett Packard, Inc. Palo Alto, CA, USA).
9
Western Blot Analysis of REV3L
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Cell lysates (100 µl; 2×106 cells) were prepared using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 2 µl protease inhibitor (Sigma-Aldrich; Merck KGaA). Briefly, the concentration of each protein sample was determined by bicinchoninic acid assay kit (Beyotime Institute of Biotechnology), and the total protein (20 μg/lane) extracted from each sample was separated by SDS-PAGE on 8% gels and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk and incubated with primary antibodies against REV3L (1:1,000; catalog no. GTX17515; GeneTex, Inc., Irvine, CA, USA) and GAPDH (1:10,000; catalog no. G8795; Sigma-Aldrich; Merck KGaA) at 4°C overnight, followed by incubation with anti-rabbit peroxidase-conjugated secondary antibody (1:80,000; catalog no. a0545; Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. Protein bands were visualized using Enhanced Chemiluminescence detection reagents (Thermo Fisher Scientific, Inc. USA). GAPDH served as a loading control.
10
Western Blot Analysis of Katanin p60
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After transfected with shRNAs or plasmids for 48 h, cells were lysed using radioimmunoprecipitation assay buffer containing 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS (0.1 ml/1×106 cells; Beyotime Institute of Biotechnology). Total protein was extracted and its concentration was measured using a BCA protein assay kit (Beyotime Institute of Biotechnology). Extracted proteins (50 µg) were separated by SDS-PAGE (5% stacking gels, 12% resolving gels) and transferred into a nitrocellulose membrane. Subsequent to blocking with 5% skim milk (GE Healthcare, Chicago, IL, USA) for 2 h at room temperature, the membranes were washed and incubated with anti-katanin p60 antibody (dilution, 1:500; Abcam, Cambridge, UK) and anti-GADPH antibody (dilution, 1:1,000; Abcam) overnight at 4°C. Then the membranes were washed with 1X TBST buffer (pH 7.6; 2.42 g/l Tris, 8 g/l NaCl, 0.5 ml Tween-20) and incubated with the peroxidase-conjugated anti-rabbit secondary antibody (dilution, 1:1,000; cat no. A0545; Sigma-Aldrich; Merck KGaA) for 90 min at room temperature, an ECL chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.) was used to visualize the proteins. Densitometry was performed using ImageJ software (version 1.46; National Institutes of Health, Bethesda, MD, USA).
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