All fractions were mixed with the equal volume of the 2X sample loading buffer (125 mM Tris-HCl, 4% (w/v) SDS, 20% (v/v) glycerol, and 0.01% (w/v) bromophenol blue) with dithiothreitol and boiled for 10 min. Mixtures were briefly centrifuged prior to loading, electrophoresed through 10% SDS-polyacrylamide gels, and electro-transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 1X TBS-T (137 mM NaCl, 27 mM KCl, 250 mM Tris-HCl, and 0.1% Tween-20) containing 5 % (w/v) skim milk for an hour at room temperature prior to incubating with an anti-GFP, anti-RFP (dilution: 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) or anti-CP antibody (CTV-908 [35 (link)]; dilution: 1:500) with 1X TBS-T for another hour. Anti-rabbit IgG conjugated to horseradish peroxidase was used as a secondary antibody (dilution: 1:20,000; Santa Cruz Biotechnology, Dallas, TX, USA), and the signal was visualized on chemiluminescence film (X-OMAT LS; Carestream Health Rochester, NY, USA) in a dark room (Carestream Kodak, Sigma-Aldrich, St. Louis, MO, USA).
Anti rabbit igg conjugated to horseradish peroxidase
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-rabbit IgG conjugated to horseradish peroxidase is a laboratory reagent used as a detection tool in various immunoassay techniques. It consists of rabbit immunoglobulin G (IgG) molecules covalently linked to the enzyme horseradish peroxidase (HRP). This conjugate can be utilized to detect and visualize the presence of rabbit-derived antibodies in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Gnrh 1 5, by Bachem (1 mentions)
Tgf βrii, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Gs 800 calibrated densitometer, by Bio-Rad (1 mentions)
Ecl regent, by Cytiva (1 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti rabbit igg conjugated to horseradish peroxidase
1
Western Blot Analysis of Fluorescent Proteins
2
Characterizing TAZ and Integrin Signaling
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The tissues were homogenized in RIPA buffer (Sigma) and the total protein concentrations of the homogenates were measured using the BCA reagent. Equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. After blocking, the membranes were incubated with antibodies against human TAZ (Abcam), human β1 integrin (Abcam), and β-actin (Cell Signalling Technology).
ME180 cells were grown on 100‐mm culture dishes until they reached 80% confluency and then incubated with su6656, an inhibitor of the Src family of kinases (1 μ M) (Calbiochem. Calif., USA), or a vehicle control (DMSO) for 1 hr. The lysates were prepared in RIPA buffer. The cytosolic and nuclear extracts were obtained using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher, Waltham, MA). Equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. After blocking, the membranes were incubated with primary TAZ antibodies (Abcam). The secondary antibody was an anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), diluted 1:2000 and incubated with the membrane for 1 h. The protein bands were detected using enhanced chemiluminescence plus detection system according to the manufacturer's instructions (BioRad).
ME180 cells were grown on 100‐mm culture dishes until they reached 80% confluency and then incubated with su6656, an inhibitor of the Src family of kinases (1 μ M) (Calbiochem. Calif., USA), or a vehicle control (DMSO) for 1 hr. The lysates were prepared in RIPA buffer. The cytosolic and nuclear extracts were obtained using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher, Waltham, MA). Equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. After blocking, the membranes were incubated with primary TAZ antibodies (Abcam). The secondary antibody was an anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), diluted 1:2000 and incubated with the membrane for 1 h. The protein bands were detected using enhanced chemiluminescence plus detection system according to the manufacturer's instructions (BioRad).
3
GnRH-(1–5) Receptor Signaling in GN11 Cells
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GnRH-(1–5) was purchased from Bachem (Torrance, CA, USA) and reconstituted to 10 mM in distilled water and stored in 12 µL aliquots at −80°C. GN11 cells (18 (link)) generously donated by Dr. Sally Radovick (Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech Inc., Herndon, VA, USA) without antibiotics and supplemented with 7% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 3% newborn calf serum (Hyclone), 25 mM glucose, and 5 mM l -glutamine (7 (link), 11 (link), 19 (link)). Cells were maintained at 37°C in an atmosphere with 5% CO2. In addition, all experiments were conducted at 37°C in an atmosphere with 5% CO2. All GN11 cells used for experimentation were below passage 25. GN11 cells originated from an olfactory tumor extracted from a male mouse (18 (link)). The antibodies used in this study were specific for the TGF-βRI (1:1,000; Cat. No. SC-9048, Santa Cruz Biotechnology, Dallas, TX, USA), TGF-βRII (1:1,000; Cat. No. SC-400, Santa Cruz Biotechnology), GAPDH (1:2,000; Cat. No. SC-25778, Santa Cruz Biotechnology), and anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000; Cat. No. 170-6515, Bio-Rad, Hercules, CA, USA).
4
Western Blot Analysis of Protein Expression
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Cells were washed and lysed in 0.15 ml cell extraction buffer (Invitrogen). Equivalent amounts of protein were solubilized in 2× SDS sample buffer, separated on 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membrane, blocked with 5% nonfat dry milk containing 0.1% Tween 20 for 1 h at room temperature, and washed thrice with wash buffer (1×TBST). Blots were incubated with primary antibodies against AP-2α, α7 nAChR at 1:1000 dilution, or EP4 (1:4000), overnight at 4°C, then washed thoroughly, and incubated with secondary anti-rabbit IgG conjugated to horseradish peroxidase (1:2,000 dilution; Santa Cruz) for 1 h at room temperature. Blots were stained by ECL regents (Amersham Life Science) and exposed to X-ray film, and proteins were quantified by densitometric scanning using a Bio-Rad GS-800 calibrated densitometer.
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