Protein extracts were resolved using 12% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Bedford, MA, USA). The PVDF membranes were blocked with WB blocking solution (Beyotime Biotechnology Co., Ltd, Zhejiang, China), and subsequently washed four times for 15 min with Tris-buffered saline with Tween-20 (TBST; Merck Millipore) at room temperature and incubated with the following primary antibodies: Rabbit anti-human p53 polyclonal antibody (cat. no. 12571), rabbit anti-human CCND1 polyclonal antibody (cat. no. 3300), rabbit anti-human cyclin-dependent kinase 4 (CDK4) polyclonal antibody (cat. no. 12790), rabbit anti-human E-cadherin polyclonal antibody (cat. no. 3195), rabbit anti-human claudin 1 (CLDN1) polyclonal antibody (cat. no. 4933), rabbit anti-human GAPDH polyclonal antibody (cat. no. 5174), all at 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA USA). Following extensive washing, membranes were incubated with a peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, Ca, USA) for 1 h. After washing four times for 15 min with TBST at room temperature, the immunoreactivity was visualized using an enhanced chemiluminescence (ECL kit, Pierce Biotechnology, Inc., Rockford, IL, USA).
Peroxidase conjugated goat anti rabbit igg secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Peroxidase-conjugated goat anti-rabbit IgG secondary antibody is a detection reagent used in immunological assays. It is a secondary antibody that binds to rabbit primary antibodies and is conjugated with the enzyme peroxidase. This allows for the detection and visualization of target proteins or antigens in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Wb blocking solution, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Direct detect spectrometer, by Merck Group (1 mentions)
Peroxidase conjugated goat anti mouse igg secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Ab124741, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab31387, by Abcam (1 mentions)
Av41970, by Merck Group (1 mentions)
Ab32096, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
4 protocols using peroxidase conjugated goat anti rabbit igg secondary antibody
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Brain Metabolic Transporters
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Brain cortex lysate was produced by sonication in 400 µL of 2% SDS containing protease inhibitors (Complete Mini EDTA-free, Roche Diagnostics GmbH, Mannheim, Germany). The lysates were centrifuged at 4 °C, 1400 × g for 5 min and the supernatants were collected. Protein content was determined by IR spectrometry (Direct Detect Spectrometer, Merck Millipore, Darmstadt, Germany). 50 µg of protein lysate were loaded onto a 4–20% Run Blue SDS gel and electrotransferred onto nitrocellulose membranes. Blots were probed with anti-MCT1 rabbit polyclonal antibody (1:200; Merck Millipore), anti-MCT2 mouse monoclonal antibody (1:200, Santa Cruz, Heidelberg, Germany), anti-MCT4 rabbit polyclonal antibody (1:200; Merck Millipore), anti-GLUT1 rabbit polyclonal antibody (1:500, Merck Millipore) or anti-GLUT3 rabbit monoclonal antibody (1:1000, Abcam, Cambridge, UK) and peroxidase conjugated goat anti-rabbit IgG secondary antibody (1:2000 for MCT1, MCT4, GLUT3; 1:4000 for GLUT1; Santa Cruz) or peroxidase conjugated goat anti-mouse IgG secondary antibody (1:4000; Santa Cruz).
3
Quantitative Western Blot Analysis of OGG1
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The cell pellets were homogenized in a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate and 1% protease inhibitor. The lysate was centrifuged at 13,000 × g for 10 min at 4°C. Protein concentrations were determined using a BCA kit (Pierce; Thermo Fisher Scientific, Inc.). The protein samples with equal amounts (300 µg) were then separated by 10% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked with 5% skim milk at 37°C and incubated with rabbit anti-human OGG1 primary antibody (1:1,000, ab124741; Abcam) and GADPH (1:2,000, ab181602; Abcam), overnight at 4°C. Peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, Inc., sc-2004; 1:10,000) was then added for a further 2-h incubation at room temperature, and the cells were then visualized using an ECL kit (Pierce; Thermo Fisher Scientific, Inc.) for protein band quantification by densitometry using ImageJ software 1.52a (NIH).
4
Protein Expression Analysis in Brain Regions
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Tissue samples from the hippocampus and prefrontal cortex of offspring were homogenized in extraction buffer (C500006, Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. Each sample was adjusted to a final protein concentration of 1 μg/μl, mixed with Laemmeli’s sample buffer, and boiled for 5 min. Samples (40 mg) were loaded onto 8% bisacrylamide gels and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrophoretically transferred from gels to polyvinylidene fluoride (PVDF) membranes that were then incubated with the following primary antibodies: anti-BDNF (1:200 dilution, AV41970, Sigma-Aldrich, St. Louis, MO, USA), anti-SERT (1:200, AG1204, Abgent, San Diego, CA, USA), anti-CREB (1:500 dilution, ab31387, Abcam, Cambridge, MA, USA), anti-pCREB (1:500 dilution, ab32096, Abcam), and anti-GAPDH (1:2500 dilution, ab9485, Abcam). Dilutions of peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:2000 dilution, sc-2004, Santa Cruz Biotechnology, Dallas, TX, USA) were prepared following the manufacturer’s instructions. Immuno-positive bands were visualized using a chemiluminescent method (G:BOX chemiXR5, SYNGEN, Sacramento, CA, USA), and the band densities were determined with the Gel-Pro32 software [39 (link)]. All western blotting experiments were repeated at least three times.
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