HEI-OC1 cells and cochleae were lysed with cold RIPA Lysis Buffer plus PMSF. A BCA Protein Quantification Kit (Thermo, #NCI3227CH) was used to measure the protein concentration with GAPDH as the reference protein in line with the manufacturer’s instructions. Cleaved-CASP3 was measured using an anti-cleaved-CASP3 antibody (CST, 9664S), cleaved-CASP9 was measured using an anti-Cleaved-CASP9 antibody (Abcam, ab202068), and GAPDH was measured using an anti-GAPDH rabbit polyclonal antibody (Google). Peroxidase-conjugated goat anti-rabbit immunoglobulin G (Santa Cruz Biotechnology) was used as the secondary antibody. The proteins were bound to polyvinylidene fluoride membranes, and a SuperSignal West Dura chemiluminescent substrate kit was used to detect the complexes according to the manufacturer’s instructions. The western blots were semi-quantified using ImageJ to measure the intensities of the target bands, and the ratio of the target protein to the control protein in each sample was calculated.
Peroxidase conjugated goat anti rabbit immunoglobulin g
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Peroxidase-conjugated goat anti-rabbit immunoglobulin G is a secondary antibody used to detect the presence of rabbit primary antibodies in various immunological assays. It is a conjugate of goat-derived anti-rabbit IgG antibodies and the enzyme horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Ab202068, by Abcam (1 mentions)
Bca protein quantification kit, by Thermo Fisher Scientific (1 mentions)
Anti actin rabbit polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti bax, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Pierce ecl chemiluminescence reagents, by Thermo Fisher Scientific (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Sc 488, by Santa Cruz Biotechnology (1 mentions)
Sc 2004, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
4 protocols using peroxidase conjugated goat anti rabbit immunoglobulin g
1
Quantifying Apoptosis Markers in HEI-OC1 Cells
2
Western Blot Analysis of Apoptosis Markers
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Cerebral tissues were sampled at 4, 12, 24 and 72 h after ischemia whereas cultured cells were collected at 24 h after OGD. Western blots were performed using protocols as previously described [17 (link)]. The following antibodies were used: rabbit anti-ASPP1 polyclonal antibody (1:200, Abbiotec, San Diego, CA, USA), rabbit anti-ASPP2 polyclonal antibody (1:200, Abbiotec), rabbit anti-iASPP polyclonal antibody (1:1000, Abcam, Hong Kong, China), rabbit anti-p53-upregulated mediator of apoptosis (Puma) (1:1000, Abcam), rabbit anti-Bax (1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), cleaved caspase-3 (1:1000, Cell Signaling Technology Inc. Beverly, MA, USA) and rabbit anti-Actin polyclonal antibody (1:2000, Santa Cruz Biotechnology Inc.). Membranes were then incubated with peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:2000, Santa Cruz Biotechnology Inc.). The blots were visualized by chemiluminescence (Millipore, Billerica, MA, USA). Protein levels were normalized to β-Actin as a loading control. The relative optical density of protein bands was measured after subtracting the film background.
3
Western Blot Analysis of K-Ras Protein
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Protein lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to the nitrocellulose membrane. After blocking, the membranes were incubated with purified rabbit anti-K-Ras antisera at 4°C overnight. The next day, the membranes were washed with phosphate-buffered saline and then incubated with peroxidase-conjugated goat anti-rabbit immunoglobulin G (Santa Cruz Biotechnology, Texas, USA). Immunodetection was conducted with Pierce® ECL chemiluminescence reagents (Thermo Fisher) and exposed on an X-ray film. β-actin was used as an internal reference for relative quantification.
4
Quantifying Hippocampal ID1 Levels in Mice
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ID1 levels in the mouse hippocampus (n=7 at each time point) were analyzed using a previously-published method (28 (link)). Hippocampal tissues were homogenized and ID1 levels were determined using a micro bicinchoninic acid (BCA) protein assay kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Nitrocellulose transfer membranes (Pall Life Sciences, Port Washington, NY, USA) were incubated with rabbit anti-ID1 (1:1,000; sc-488; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), β-actin (1:2,000, sc-47778; Santa Cruz Biotechnology, Inc.) and exposed to peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:2,000; sc-2004; Santa Cruz Biotechnology, Inc.) for 2 h at 4°C and an enhanced luminol-based chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). The results of this analysis were scanned and densitometric analysis, as the relative optical density (ROD), was used for quantification of the bands using Scion Image software, version 2.0 (Scion Corporation, Frederick, MD, USA). A ratio of the ROD was calibrated as a percentage, with control mice designated as 100%.
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