Total protein was extracted from the cells or tissues using lysis buffer containing protease inhibitors. Proteins (30 µg) from each sample were loaded on 10% SDS-polyacrylamide gels for electrophoresis. Following electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes (GE Healthcare, Chalfont, UK), which were then blocked with 10% non-fat milk in Tween/Tris-buffered salt solution (TTBS; 20 mm Tris-Cl, pH 7.5, 0.15 M NaCl and 0.05% Tween-20) for 1 h. Following incubation with the anti-BLM antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight, the membrane was washed and incubated with IRDye® 800CW donkey anti-goat IgG secondary antibody at room temperature for 2 h followed by LI-COR Odyssey gel imaging scanner detection (LI-COR Biosciences, Lincoln, NE, USA). To verify equal loading of protein, the blots were reprobed with primary monoclonal antibody against β-actin (ProteinTech Group, Inc., Chicago, IL, USA).
Irdye 800cw donkey anti goat igg secondary antibody
Manufactured by LI COR
Sourced in United States
The IRDye® 800CW donkey anti-goat IgG secondary antibody is a near-infrared fluorescent dye-labeled antibody. It is designed to detect and quantify goat primary antibodies in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Anti blm antibody, by Santa Cruz Biotechnology (1 mentions)
Primary monoclonal antibody against β actin, by Proteintech (1 mentions)
Protease inhibitor cocktail tablet, by Merck Group (1 mentions)
Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (1 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Pageruler plus prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
2 protocols using irdye 800cw donkey anti goat igg secondary antibody
1
Western Blot Analysis of BLM Protein
2
Western Blot Analysis of FAN1 Protein
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Cells were washed once in DPBS and lysed with RIPA buffer (Sigma-Aldrich) containing cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets (Merck). Protein samples were denatured at 70 °C for 10 minutes in 4× NuPAGE LDS Sample Buffer (Life Technologies). Next, 40 µg of cell protein extract per sample was separated on NuPAGE 4–12% Bis-Tris gradient gels (Life Technologies) alongside PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) using MOPS SDS NuPAGE Running Buffer (Invitrogen) at 200 V for 50 minutes. Where purified proteins were run, 30 ng of protein per lane was loaded. Gels were then transferred to methanol-activated Immobilon-P PVDF membranes (Sigma-Aldrich) using NuPAGE Transfer Buffer (Invitrogen) at 120 V for 45 minutes. The membrane was blocked in 5% milk in PBS-T and incubated overnight at 4 °C with anti-FAN1 (CHDI, sheep polyclonal, 1:1,000) and anti-β-tubulin (UpState, 05-661, mouse monoclonal, 1:10,000). Donkey anti-mouse IgG Alexa Fluor 680 (Invitrogen, A32788, 1:10,000) and IRDye 800CW donkey anti-goat IgG secondary antibody (LI-COR Biosciences, 926-32214, 1:15,000) were used as secondary antibodies. Immunoblots were visualized with the Odyssey CLx Imaging System using β-tubulin as a loading control.
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