ELISA (enzyme-linked immunosorbent assay) was utilized to measure expression of full-length SosV-RBP and NDV-RBP on HEK293T cells (91 (link)). Lipofectamine-2000 transfected cells were washed with PBS pH 7.4 following 18 h incubation at 37 °C, 5% CO2, counted and seeded into an ELISA plate at a density of 25,000 cells per mL. Cells were bound overnight at 4 °C prior to fixation for 15 min in 4% paraformaldehyde. Following thorough washing, cells were blocked in PBS-5% milk for 1 h and subsequently reacted with rabbit anti-6xhis-tag antibody (Abcam, product no. ab9108) for 1 h at room temperature. Cells were repeatedly washed prior to addition of horseradish peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories, product no. PI-1000), for 1 h at 21 °C. Cells were repeatedly washed prior to addition of TMB substrate kit (Pierce). Stop solution (2 M sulphuric acid) was added following a 15-min incubation at room temperature, and absorbance was read at 430 nm using a using an Infinite F200 plate reader (TECAN).
Horseradish peroxidase conjugated goat anti rabbit igg
Manufactured by Vector Laboratories
Sourced in United States
Horseradish peroxidase-conjugated goat anti-rabbit IgG is a secondary antibody used in various immunoassay techniques. It binds to rabbit primary antibodies and is conjugated to the enzyme horseradish peroxidase, which can be used for signal detection and amplification.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (4 mentions)
Sodium orthovanadate, by Fujifilm (3 mentions)
Sds page, by Fujifilm (3 mentions)
Complete edta free, by Merck Group (3 mentions)
Clarity western enhanced chemiluminescence ecl substrate, by Bio-Rad (3 mentions)
Phospho akt ser473, by Cell Signaling Technology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Protein quanti cation kit rapid, by Dojindo Laboratories (2 mentions)
Rabbit anti 6xhis tag antibody, by Abcam (1 mentions)
Infinite f200 plate reader, by Tecan (1 mentions)
Radioimmunoprecipitation assay (ripa), by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg, by Vector Laboratories (1 mentions)
Enhanced chemiluminescence reagent, by Promega (1 mentions)
β actin, by Merck Group (1 mentions)
Anti actin antibody, by Cell Signaling Technology (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Protein quantification kit rapid, by Dojindo Laboratories (1 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
8 protocols using horseradish peroxidase conjugated goat anti rabbit igg
1
Quantification of Protein Expression in HEK293T Cells
2
Western Blot Analysis of ER Stress Markers
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Cells or retina tissue were lysed in radioimmunoprecipitation assay lysis buffer (RIPA, Santa Cruz). Western blot analysis was performed as described previously [4 (link),5 (link)]. The following primary antibodies were used at the indicated dilutions: anti-phospho-eIF2α (1:1,000), anti-eIF2α (1:1,000), anti-ATF4 (1:500), anti-CHOP (1:500), anti-phospho-JNK (1:500), anti-phospho-p38 (1:1000), antiphospho-p44/42(1:1000), anti-phospho-NF-κB (1:1000), and anti-β-actin (1:5,000) antibodies. Horseradish peroxidase-conjugated goat-anti-rabbit IgG (Vector Laboratories, Bulingame, CA) and goat anti-mouse IgG (Vector Laboratories) were used as secondary antibodies.
3
Western Blot Analysis of Protein Samples
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Western blot analysis was performed as previously described [44 (link)]. Briefly, protein samples (30 μg protein/lane) were separated by 12.5 % sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and then incubated with 3D5 mouse monoclonal antibody (1: 5000) overnight at 4°C. This was followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1: 5000; Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Immunoreactivity was detected using enhanced chemiluminescence reagent (Promega, Madison, WI, USA). β-Actin (1: 5000, Sigma–Aldrich, MO, USA) was used as a loading and internal control to enable sample normalization.
4
Western Blot Analysis of PKCα Phosphorylation
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Equal amounts of proteins (20 µg/well) were separated by sodium dodecyl–sulfate polyacrylamide gel electrophoresis (Wako) and transferred to a membrane using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membrane was washed three times with Tween-phosphate-buffered saline (PBS) (1×) and incubated with 5% skimmed milk in Tween-PBS (1×) for 60 min at room temperature to block nonspecific binding. The membrane was immunoblotted with anti-phosphoPKCα (Ser657; Abcam, Cambridge, UK) or anti-ß-actin antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) at 4 °C overnight. The reacted proteins were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000; Vector Laboratories, Burlingame, CA, USA) for 60 min at room temperature and visualized by chemiluminescence using the Clarity Western ECL Substrate (Bio-Rad).
5
Extracellular Vesicle Protein Analysis
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Tissue and HUVECs were homogenized in ice-cold Pierce RIPA Buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitor cocktail cOmplete EDTA-free (Sigma-Aldrich), phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich), and sodium orthovanadate (Wako, Tokyo, Japan). Protein concentration was determined using a Protein Quantification Kit-Rapid (Dojindo Laboratories). Equal amounts of proteins were separated via SDS-PAGE (Wako) and transferred to a membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skimmed milk and immunoblotted with primary antibodies that recognize one of the following targets at 4 °C overnight: CD63 (Abcam), CD9 (Abcam), CD81 (Cell Signaling Technology, Inc. Danvers, MA, USA), β-actin (Cell Signaling Technology), phospho-Akt (Ser473)(Cell Signaling Technology), Akt (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology), and caspase-3 (Cell Signaling Technology). Signals were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) and visualized using chemiluminescence with the Clarity Western Enhanced chemiluminescence (ECL) substrate (Bio-Rad). ECL signals were digitized using ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA).
6
Protein Isolation and Western Blot Analysis
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Tissue and HUVECs were homogenized in ice-cold Piece RIPA Buffer (Thermo Fisher Scienti c) containing protease and phosphatase inhibitor cocktail cOmplete EDTA-free (Sigma-Aldrich), phenylmethylsulfonyl uoride (PMSF) (Sigma-Aldrich), and sodium orthovanadate (Wako, Tokyo, Japan). Protein concentration was determined using a Protein Quanti cation Kit-Rapid (Dojindo Laboratories). Equal amounts of proteins were separated via SDS-PAGE (Wako) and transferred to a membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skimmed milk and immunoblotted with primary antibodies that recognize one of the following targets at 4 °C overnight: CD63(Abcam), CD9(Abcam), CD81(Cell Signaling Technology, Inc. Danvers, MA, USA), βactin (Cell Signaling Technology), phospho-Akt (Ser473) (Cell Signaling Technology), and Akt (Cell Signaling Technology). Signals were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) and visualized using chemiluminescence with the Clarity Western Enhanced chemiluminescence (ECL) Substrate (Bio-Rad). ECL signals were digitized using ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA).
7
Protein Expression and Signaling Assay
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Tissue and HUVECs were homogenized in ice-cold Pierce RIPA Buffer (Thermo Fisher Scienti c) containing protease and phosphatase inhibitor cocktail cOmplete EDTA-free (Sigma-Aldrich), phenylmethylsulfonyl uoride (PMSF) (Sigma-Aldrich), and sodium orthovanadate (Wako, Tokyo, Japan). Protein concentration was determined using a Protein Quanti cation Kit-Rapid (Dojindo Laboratories).
Equal amounts of proteins were separated via SDS-PAGE (Wako) and transferred to a membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skimmed milk and immunoblotted with primary antibodies that recognize one of the following targets at 4 °C overnight: CD63 (Abcam), CD9 (Abcam), CD81 (Cell Signaling Technology, Inc. Danvers, MA, USA), βactin (Cell Signaling Technology), phospho-Akt (Ser473)(Cell Signaling Technology), Akt (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology), and caspase-3 (Cell Signaling Technology). Signals were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) and visualized using chemiluminescence with the Clarity Western Enhanced chemiluminescence (ECL) substrate (Bio-Rad). ECL signals were digitized using ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA).
Equal amounts of proteins were separated via SDS-PAGE (Wako) and transferred to a membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skimmed milk and immunoblotted with primary antibodies that recognize one of the following targets at 4 °C overnight: CD63 (Abcam), CD9 (Abcam), CD81 (Cell Signaling Technology, Inc. Danvers, MA, USA), βactin (Cell Signaling Technology), phospho-Akt (Ser473)(Cell Signaling Technology), Akt (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology), and caspase-3 (Cell Signaling Technology). Signals were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) and visualized using chemiluminescence with the Clarity Western Enhanced chemiluminescence (ECL) substrate (Bio-Rad). ECL signals were digitized using ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA).
8
Western Blot Analysis of Spinal Cord
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The spinal cords of four rats in each group were removed and transversely cut at the lumbar enlargement (L2-L5) [19] , followed by homogenization and centrifugation. The supernatants were proportionally diluted with 3× loading buffer and heated for 15 min at 37 °C. The samples were loaded onto 12% Tris/tricine SDS gels, transferred to PVDF membranes, and blocked with 5% milk solubilized in TBST (pH 7.4, 10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20) at room temperature for 2 h. Subsequently, the membranes were incubated with rabbit anti-BDNF monoclonal antibody (1:1000; Abcam), rabbit anti-TrkB polyclonal antibody (1:1000; Abcam), rabbit anti-PSD-95 monoclonal antibody (1:1000; Abcam), rabbit anti-SYP monoclonal antibody (1:1000; Millipore), or GAPDH (1:1000; Bioworld, Louis Park, MN, USA) at 4 °C overnight, followed by incubation with horseradish peroxidaseconjugated goat anti-rabbit IgG (1:2000, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The immunoreactive bands were detected using the ECL plus detection system. GAPDH served as a loading control.
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