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Goat anti mouse igg conjugated to horseradish peroxidase

Manufactured by Santa Cruz Biotechnology
Sourced in United States

Goat anti-mouse IgG conjugated to horseradish peroxidase is a secondary antibody that binds to mouse primary antibodies. The horseradish peroxidase enzyme is used as a reporter molecule for detection in various immunoassay techniques.

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6 protocols using goat anti mouse igg conjugated to horseradish peroxidase

1

Western Blot Analysis of ER Stress Markers

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The MSCs were lysed using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) to obtain total cellular protein. Cell lysates (20 µg of total protein) were separated on 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to the nitrocellulose membrane. After the blots had been washed with TBST (10 mM Tris-HCl (pH 7.60), 150 mM NaCl, 0.05% Tween-20), they were blocked with 5% skimmed milk for 1 h and incubated with appropriate primary antibodies at the dilutions recommended by the supplier. Antibodies against GRP78, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, IRE1α, CHOP, p-IRE1α, NF-κB, p-NF-κB, JNK, p-JNK, p38, p-p38, BCL-2, BAX, cleaved caspase-3, cleaved PARP-1, PrPC, MnSOD, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The membranes were then washed, and primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
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2

PA-D4 Protein Expression Analysis

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To confirm the expression of PA-D4, 293T cells were plated at 1×106 cells per well into six-well plates. Cells were transfected with 1 μg of plasmid DNA using the transfection agent Vivagen (Vivagen, Seoul, Korea) according to the manufacturer’s guidelines. After 48 h, cells were harvested and lysed. The cell lysates were separated by 4–20% polyacrylamide gel electrophoresis. Proteins were then transferred from the gel to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h in 5% nonfat dry milk in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) and probed for 1.5 h with primary mouse anti-PA-D4 sera diluted 1:5,000 in TBST/5% nonfat skim milk. Membranes were then washed with TBST 3 times and incubated for 1 h with goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotech, Santa Cruz, USA) at a 1:5,000 dilution in TBST. After additional 4 washes with TBST, the bound antibody was visualized using Western Lighting Plus-ECL (PerkinElmer, Waltham, USA).
We also constructed plasmids that expressed PA-D4 antigens as fusion proteins with enhanced green fluorescent protein as described above. Expression of EGFP was determined using a confocal laser-scanning microscope (LSM 5; Carl Zeiss, Oberkochen, Germany) at 48 h after transfection.
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3

Unfolded Protein Response Pathway Analysis

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Protein extracts were separated via 8% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membrane. After blocking membranes with 5% skim milk for 1 h, glucose-regulated protein 78 (GRP78) primary antibodies against PrPC, protein kinase-like endoplasmic reticulum kinase (PERK), phospho-PERK (p-PERK), inositol-requiring enzyme 1α (IRE1α), p-IRE1α, activating transcription factor 4 (ATF4), and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) were incubated overnight. After washing with PBS, the membranes were incubated with goat anti-rabbit immunoglobulin g (IgG) or goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, England, UK).
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4

Western Blot Analysis of UPR Markers

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The MSC homogenates (20 μg protein) were separated via 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to nitrocellulose. After the blots had been washed with TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.05% Tween-20), the membranes were blocked with 5% skim milk for 1 h and incubated with the appropriate primary antibodies at the dilutions recommended by the supplier. Antibodies against GRP78, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, IRE1α, p-IRE1α, JNK, p-JNK, p38, p-p38, CHOP, BCL-2, Bax, cleaved caspase-3, cleaved PARP-1, Akt, phosphor-Akt, PrPC, α-tubulin, and β-actin were all purchased from Santa Cruz Biotechnology. The membranes were then washed, and the primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, England, UK).
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5

Protein Expression Analysis of MSCs

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MSCs homogenates were extracted by using RIPA lysis buffer (Thermo Scientific). Cell lysates (20 μg of total protein) were separated by using 6–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose membrane (Thermo Scientific). After the blots had been washed with a solution comprising 10 mM Tris-HCl (pH 7.6; Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), and 0.05% Tween-20 (Sigma-Aldrich), the membranes were incubated with 5% bovine serum albumin for 1 h at room temperature and then, incubated with appropriate primary antibodies against phosphoinositide 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), Akt, phosphorylated Akt (p-Akt), phosphorylated 5′-AMP-activated protein kinase (AMPK), mechanistic target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), LC3, beclin 1, ATG7, p62/Sequestosome 1 (p62/SQSTM1), senescence marker protein 30 (SMP30), and β-actin (all from Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were then washed more than twice, and the primary antibodies were detected using a goat anti-rabbit IgG or a goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
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6

Western Blot Analysis of ROCK1 Protein

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Seventy-two hours after transfection, cells were washed with PBS and lysed in RIPA buffer containing protease inhibitor. Protein concentration was determined by BCA (Pierce, Rockford, IL, USA) assay. Total proteins (20 μg) were resolved by 10% SDS polyacrylamide gels and immobilized on PVDF membranes (Millipore, Billerica, MA, USA). Membranes were then blocked with 5% nonfat dried milk in Tris-buffered saline/0.1% Tween (TBST) and incubated overnight at 4°C with the following primary antibodies: mouse anti-human monoclonal ROCK1 antibody (sc-365628; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-human monoclonal GADPH antibody (sc-365062; Santa Cruz Biotechnology). Membranes were then washed with TBST and incubated for 1 h with goat anti-mouse IgG conjugated to horseradish peroxidase (1:5,000; Santa Cruz Biotechnology). The bands were visualized using ECL Western Blot Detection kit (Amersham Life Sciences, Piscataway, NJ, USA). GADPH was used to ensure equivalent protein loading.
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