Cells were lysed with M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific) supplemented with 1X protease inhibitor cocktail (Roche), 0.2 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride (PMSF). BCA assay (Santa Cruz) was used for protein quantification. Cell lysates or IP elutes were subjected to SDS/PAGE followed by either conventional wet transfer or dry transfer using iBlot® Gel Transfer Device (Invitrogen). Membranes were incubated with antibodies as indicated and exposed to secondary HRP-conjugated antibodies (Millipore).
Hrp conjugated antibodies
Manufactured by Merck Group
Sourced in United States
HRP-conjugated antibodies are laboratory reagents used in various immunoassay techniques. They consist of an antibody molecule covalently linked to the enzyme horseradish peroxidase (HRP). The HRP enzyme can catalyze a colorimetric reaction, allowing for the detection and quantification of target analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Cortactin, by Santa Cruz Biotechnology (2 mentions)
Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Santa Cruz Biotechnology (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Supersignal chemiluminescent hrp substrate, by Thermo Fisher Scientific (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Twist, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Cyclin d1, by Santa Cruz Biotechnology (1 mentions)
Seeblue plus2, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Mark12, by Thermo Fisher Scientific (1 mentions)
Mini protean 3, by Bio-Rad (1 mentions)
Hybond p, by Cytiva (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
10 protocols using hrp conjugated antibodies
1
Protein Extraction and Western Blotting
2
Characterization of CSP-dS VLPs
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CSP-dS VLPs and full-length recombinant CSP (rCSP) were prepared under reducing conditions, separated by SDS-PAGE using 4-12% Bis-Tris gels (NuPAGE, Thermo Fisher Scientific) and transferred onto nitrocellulose membranes using the iBlot system (Thermo Fisher Scientific) according to the manufacturer instructions. Membranes were blocked with 10% (w/v) skim milk in phosphate buffered saline (PBS) and probed with polyclonal rabbit anti-CSP IgG (1 μg/mL) and a dS-specific mouse monoclonal antibody (7C12; 1/1000). This was followed by species-specific detection HRP-conjugated antibodies (1/5000; Millipore, USA). Protein bands were detected using SuperSignal Chemiluminescent HRP substrate (Thermo Fisher Scientific) and imaged using the ChemiDoc System (Bio-Rad, USA).
3
Western Blot Analysis of Protein Expression
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Following starving and treatment, cells were harvested, lysed with 2X Laemmli sample buffer, and boiled for 10 min. SDS electrophoresis was performed on 7.5% polyacrylamide gels run at 120 mA (RT for 45 min). Proteins were then transferred to nitrocellulose membranes (Biorad Laboratories, Hercules, CA) at 350 mA at RT for 2 h. Residual binding sites were blocked for 1 h at RT in TBST/3% low‐fat milk. Membranes were washed three times with TBST and incubated over night at 4°C with the following primary antibodies (1 μg/mL): p27, Cyclin D1, Twist, Tubulin (all from SantaCruz Biotechnology Inc. Heidelberg, Germany), Gro‐α (Abcam, Cambridge, UK).
After incubation with anti‐mouse or anti‐rabbit secondary HRP‐conjugated antibodies (1:2000 and 1:10000, respectively, from Sigma), membranes were washed and treated with enhanced LumiGLO chemiluminescence reagents (KPL, Gaithersburg, Maryland, USA) before exposure to X‐ray film.
Following acquisition using a CCD camera in a light table with shading correction, densitometric analysis was performed by ImageJ 1.38 (Windows version of NIH Image, (http://rsbweb.nih.gov/ij/ ) and background correction was done with the default settings (rolling ball radius = 50).
After incubation with anti‐mouse or anti‐rabbit secondary HRP‐conjugated antibodies (1:2000 and 1:10000, respectively, from Sigma), membranes were washed and treated with enhanced LumiGLO chemiluminescence reagents (KPL, Gaithersburg, Maryland, USA) before exposure to X‐ray film.
Following acquisition using a CCD camera in a light table with shading correction, densitometric analysis was performed by ImageJ 1.38 (Windows version of NIH Image, (
4
Immunoblotting Analysis of Protein Samples
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Cell samples were homogenized and solubilized in standard 2X Laemmli buffer (Invitrogen) supplemented with complete protease inhibitor cocktail (Roche). Following centrifugation at 20,000 g for 30 minutes, the concentration of supernatant proteins was analyzed using the Bio-Rad DC Protein Assay Kit according to the manufacturer's protocol. Extracted proteins were subjected to SDS-PAGE (Mini-Protean-III, Bio-Rad), stained with Coomassie or blotted onto PVDF membranes (Hybond-P, Amersham Bioscience), and probed with mouse monoclonal anti-Myc or anti-FLAG antibodies (Sigma). Molecular weight (MW) standards (MARK-12 and SeeBlue Plus2 from Invitrogen) were included on each gel. Equivalence of protein loading was confirmed by Amido Black staining of blots after immunodetection. Blocking, washing, incubation with diluted primary and secondary HRP-conjugated antibodies (Sigma), and visualization of immunodecorated bands by the Super-Signal West Pico PLUS chemiluminescent substrate (Thermo Scientific) were carried out as previously described [19 (link)].
5
Huh-7 and HepG2 Cell Lysate Analysis
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Whole cell lysates were obtained from Huh-7 and HepG2 cell lines with or without transfection with pSVL and pSVM and 10 nM thapsigargin after 72 hours, and further processed by SDS-Page followed by western blotting, as previously described [52 (link)]. Immunodetection was performed with primary antibodies against BiP, (Abcam) and β-actin (Sigma-Aldrich). Secondary HRP-conjugated antibodies (Sigma-Aldrich) were detected by incubating the immunoblots with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Thermo Fisher Scientific). The luminescent reactivity was measured by using Fusion image capture and further quantified with Bio1D analysis system (PEQLAB Biotechnologie GmbH). Anti β-actin was used as equal loading control and protein quality.
6
Evaluation of ZIKV Antigen Expression in MVA
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To test the expression of the ZIKV antigens present in MVA-ZIKV, monolayers of DF-1 cells were mock infected or infected at 5 PFU/cell with MVA-WT, MVA-Δ-GFP, or MVA-ZIKV. At 24 hpi cells extracts were loaded and separated in 10% SDS-PAGE, and then analyzed by Western blotting with a rabbit polyclonal antibody against ZIKV prM (Genentex; diluted 1:2,000) or a mouse monoclonal antibody against ZIKV E (BioFront Tech; diluted 1:5,000) to detect the ZIKV prM and E proteins. As loading controls, a rabbit anti-β-actin antibody (Cell Signaling; diluted 1:1,000), and a rabbit anti-VACV E3 antibody (Centro Nacional de Biotecnología; diluted 1:1,000) were used. Anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies (Sigma; diluted 1:2,000 or 1:5,000, respectively) were used as secondary antibodies. The immune complexes were detected with an HRP-luminol enhanced-chemiluminescence system (ECL Plus, GE Healthcare).
7
Evaluating Molecular Changes in LLC Cells
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LLC cells were seeded into 100 mm Petri dish and incubated for 24 h (70–80% confluency). Cells were treated with C60 and Ber separately or C60-Ber nanocomplexes in 10 µM Ber-equivalent concentration for 24 h. Then, protein lysates were prepared as described previously [33 (link)]. A total of 30 μg of protein was separated on 10% polyacrylamide gel and transferred to nitrocellulose membrane. Primary antibodies (monoclonal anti-E-cadherin (Cell Signaling, Cell Signaling, Danvers, MA, USA), anti-vimentin (Sigma-Aldrich, St. Louis, MI, USA), anti-β-actin antibodies (Sigma-Aldrich, St. Louis, MI, USA), and polyclonal anti-Ruk/CIN85 antibody [34 (link)]) and corresponding secondary HRP-conjugated antibodies (Sigma-Aldrich, St. Louis, MI, USA) were used for Western-blot analysis. The enhanced chemiluminescence kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for detection. Semi-quantitative analysis was performed by densitometry using Gel-Pro Analyzer 3.0 (Media Cybernetics, L.P., Rockville, MD, USA).
8
Western Blot Analysis of Cell Signaling
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Following transduction, ECs were washed twice with PBS and cells were lysed in 1× LDS sample buffer (Thermo Fisher Scientific, Waltham, USA). Cell lysates were centrifuged at 12,000 r/min and protein was estimated in supernatants. Equal amount of protein was loaded onto 4–12% Bis Tris gels (Thermo Fisher Scientific, Waltham, USA), transferred to PVDF membranes, blocked for 1 h using blotting grade blocker non-fat dry milk (Bio-Rad, Hercules, USA). Primary antibodies used were EVL (Santa Cruz Biotechnology, Dallas, USA), FAK (CST, Danvers, MA, USA), FAK-Y397 (CST, Danvers, MA, USA), MLC2 (CST, Danvers, MA, USA), pMLC2-T18/S19 (CST, Danvers, MA, USA), FAK-Y576 (Abcam, Cambridge, MA, USA) and vinculin (MilliporeSigma, St. Louis, MO, USA) and imaging was carried out using a ChemiDoc MP imaging system (Bio-Rad, Hercules, USA). β-actin was visualized using HRP-conjugated antibodies (MilliporeSigma, St. Louis, MO, USA).26 (link)
9
Western Blot Analysis of Lung Proteins
10
Western Blot Analysis of Lung Proteins
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Western blotting of lung tissue and cell extract proteins was performed with densitometric analysis normalized to β-actin expression as previously reported [21 ]. Primary antibodies used in this study include EVL and cortactin (Santa Cruz Biotechnology, Dallas, USA), MLC2 and p-MLC2-T18/S19 (CST, Danvers, MA, USA), and imaging was carried out using a ChemiDoc MP imaging system (Bio-Rad, Hercules, USA). β-actin was visualized using HRP-conjugated antibodies (MilliporeSigma, St. Louis, MO, USA).
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