Human MSCs were preconditioned under 1% O2 for 3 days or maintained in ambient air as stated above. Spheroids were then formed for 48 hrs under ambient air and collected immediately. For comparison with MSCs in monolayer culture, MSCs were seeded at 5 × 104 cells/cm2 in well plates and allowed to attach overnight. Cells were then moved to 1% O2 for 3 days of preconditioning and then placed under ambient air for an additional 48 hrs before collection. Samples were collected in protein collection buffer (0.1% Triton-X, 1% Tris-EDTA, 1% protease inhibitor cocktail), and protein concentration was measured using the Pierce BCA protein assay (Thermo Fisher Scientific). 30 μg of protein per sample was resolved 10% Tris–HCl acrylamide gels and transferred onto 0.2 mm nitrocellulose. Blots were blocked in 5% nonfat milk in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 hr and probed overnight at 4°C with mouse anti-human polyclonal antibody to HIF-1α (1:500 in blocking buffer; BD Biosciences, San Jose, CA) and the loading control, rabbit anti-human β-tubulin (1:1000; #2128P; Cell Signaling, Danvers, MA). Membranes were washed and probed with horseradish peroxidase-conjugated secondary antibodies (1:1000; BD Biosciences and Cell Signaling) and reactive bands were visualized using enhanced chemiluminescence and the Bio-Rad Gel Imaging System.
Horseradish peroxidase conjugated secondary antibody
Manufactured by BD
Sourced in United States, United Kingdom
Horseradish peroxidase-conjugated secondary antibodies are laboratory reagents used in various immunoassay techniques. They consist of a secondary antibody that is chemically conjugated with the enzyme horseradish peroxidase. The horseradish peroxidase enzyme can catalyze a colorimetric or chemiluminescent reaction, which allows for the detection and visualization of target proteins or molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Anti ucp2, by Santa Cruz Biotechnology (1 mentions)
Anti vdac1, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by PerkinElmer (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Dexamethasone (dex), by Bio-Techne (1 mentions)
Ndrg1, by Cell Signaling Technology (1 mentions)
Myosin vb, by Novus Biologicals (1 mentions)
Amplex red glucose assay kit, by Thermo Fisher Scientific (1 mentions)
Phosstop easypack, by Roche (1 mentions)
Anti sglt1, by Abcam (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Amersham ecltm prime western blotting detection reagent, by GE Healthcare (1 mentions)
8 protocols using horseradish peroxidase conjugated secondary antibody
1
Hypoxic Preconditioning and MSC Spheroid Analysis
2
Western Blot Analysis of UCP2 and VDAC1
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Cells were harvested and total cell lysates prepared using lysis buffer [20 mM Tris–HCl (pH 7.2), 2 mM EGTA, 5 mM EDTA, 500 μM sodium orthovanadate, 10 mM sodium fluoride, 1% Triton X-100, 0.1% SDS and protease inhibitor cocktail]. Protein concentrations were determined using protein assay reagents (Bio-Rad, Hercules CA, USA). Forty to sixty micrograms of protein lysate was analyzed by SDS-polyacrylamide gel electrophoresis. After transfer of the proteins from the gel to a nitrocellulose membrane (Amersham Pharmacia Biotech, Freiburg, Germany), the membranes were blocked for 1 h at room temperature in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS-T) and 5% nonfat dry milk, then were incubated with anti-UCP2 (Santa Cruz Bio. Inc., MA USA) or anti-VDAC1(Abcam, London, UK) polyclonal antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (pharMingen, San Diego CA, USA). The immunoreactive bands were visualized using an enhanced chemiluminescence kit (Perkin-Elmer Life Sciences, Boston, MA, USA) and analyzed by QUANTITY ONE (Bio-Rad Hercules CA, USA). β -actin was used as the internal control.
3
Western Blot Analysis of GAPDH and TRIP6
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Cells were lysed on ice using 1X SDS lysis buffer [100 mM 2-ME, 50 mM Tris-HCl (pH 6.8), 2% w/v SDS, 10% glycerol]. Protein concentration was determined using the bicinchoninic acid method. Protein (30 µg) was separated by 10–12% SDS-PAGE and transferred to nitrocellulose membrane (GE Healthcare Life Sciences). Membranes were probed with anti-GAPDH antibody (cat. no. #sc-25778; 1:2,000; Santa Cruz Biotechnology, Inc.) and anti-TRIP6 antibody (cat. no. 612254; 1:500; BD Bioscience) overnight at 4°C and then incubated with the horseradish peroxidase-conjugated secondary antibodies (cat. no. 1662408; 1:3,000; Bio-Rad Laboratories Inc., Hercules, CA, USA) at 37°C for 1 h. The bands were visualized with the ChemiDoc XRS system (Bio-Rad Laboratories, Inc.).
4
Immunoblot Analysis of Cellular Signaling
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The protease inhibitors used for lysate preparation for the immunoblots, PhosSTOP EASYpack (#04906837001), and complete EDTA-free (#11873580001) were purchased from Roche. Dexamethasone (#1126) was purchased from Tocris Bioscience. Antibodies against phospho-NDRG1Thr346 (#5482), NDRG1 (#5196), phospho-PDK1Thr346 (#3061), PDK1 (#3062), and GAPDH (#8884) were purchased from Cell Signaling. Antibodies against Phospho-PI3K p85Tyr458/p55Tyr199 (#PA5-17387), GLUT2 (#PA5-77459), Phospho-SGK1Thr256 (#44-1260G), and Amplex™ Red Glucose Assay kit (#A22189) were purchased from Thermo Fisher Scientific, Waltham, MA, USA. Additional antibodies: CFTR (AME4991, affinity purified polyclonal antibody raised against rat CFTR-produced by Dr. Ameen), NHE3-3H3 (#MABN1813) and SGK1 (#07-315) from EMD Millipore (Burlington, MA, USA), Phospho-PKCιThr563 (iota; #GTX25813) from GeneTex (San Antonio, TX, USA), Anti-SGLT1 (#ab14686) from Abcam (Cambridge, UK), and Myosin VB (#NBP1-87746) from Novus Biologicals (Centennial, CO, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from BD Bioscience (San Jose, CA, USA). Other reagents used in this study including tamoxifen (#T9262) were purchased from Sigma Aldrich (Burlington, MA, USA).
5
Quantifying Exosomal CAR Binding to Antigens
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To test the binding of exosomal CAR to antigen, 100 μl of exosome samples of different concentrations was captured onto recombinant antigen (EGFR or HER2)-coated 96-well ELISA plates by overnight incubation at 4 °C. Next, 100 μl of 4 μg/ml anti-MYC antibody was added and incubated for 2 h at room temperature. Cetuximab and trastuzumab were added as indicated, at a concentration of 10 μg/ml. A total of 100 μl per well of horseradish peroxidase-conjugated secondary antibody (BD Biosciences) diluted in PBS containing 0.1% BSA was then added and incubated for 1 h at room temperature. The plates were developed with tetramethylbenzidine (Pierce), and the reactions were stopped using 0.5 N H2SO4. The plates were read at 450 nm using a BioTek plate reader. Recombinant MYC-tagged scFv protein directly coated onto the plates was used as the positive control.
6
Western Blotting for Porphyromonas gingivalis Virulence Factors
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Sample preparation, separation by SDS-PAGE, electro-blotting onto polyvinylidene difluoride or nitrocellulose membranes and blocking were performed as described previously53 (link). RgpA and RgpB catalytic domains were detected with rabbit polyclonal anti-Rgp antibodies; Kgp with mouse monoclonal anti-Kgp antibodies; PPAD with rabbit polyclonal anti-PPAD antibodies; and PorZ with mouse polyclonal anti-PorZ antibodies (see also above). In addition, oligohistidine-tagged PorZ was detected using mouse monoclonal THETM His-Tag antibodies (Genscript, USA) at 0.125 μg/ml. Development with polyclonal anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody (BD Pharmingen and Amersham Pharmacia) was carried out using the ECL Western Blotting substrate kit according to the manufacturer’s instructions (Pierce, UK). Streptavidin conjugated to horseradish peroxidase was used to detect MmdC, a biotinylated IM-associated protein.
7
Protein Extraction and Immunoblotting Protocol
8
Actin Cytoskeleton Protein Expression
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