Atrial samples were homogenized on ice, centrifuged (13,000g at 4°C for 15 min), and immunoabsorbed with anti-nNOS or anti-dystrophin antibodies (NCL-DYS1). Immune complexes were precipitated for 3 hours by the addition of Protein A/G–conjugated agarose (Santa Cruz) and immunoblotted with anti-nNOS and anti-dystrophin antibodies; agarose beads with immunoglobulin (Ig) G/A alone were used as control for nonspecific binding.
Protein a g conjugated agarose
Manufactured by Santa Cruz Biotechnology
Protein A/G-conjugated agarose is a chromatography matrix that comprises agarose beads conjugated with recombinant protein A and protein G. This matrix is designed for the purification of antibodies from various biological samples.
Automatically generated - may contain errors
4 protocols using protein a g conjugated agarose
1
Immunoprecipitation of nNOS and Dystrophin
2
Immunoprecipitation and Immunoblotting Analysis
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Cells were collected and treated with lysis buffer (150 mM NaCl, 50 mM Tris-Cl, 1% Triton X-100, protease inhibitor cocktail, pH 7.4). Supernatants were collected by centrifugation (15,000g, 15 min, 4 °C), and incubated with the indicated antibodies (1 μg ml−1) for 6 h at 4 °C, followed by immunoprecipitation with 20 μl protein A/G conjugated agarose (Santa Cruz Biotechnology). The precipitates were completely washed with lysis buffer and detected through immunoblotting.
3
Immunoprecipitation and Western Blot for TNIK
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Whole-cell lysates were harvested with RIPA buffer (Cell signaling) and the concentrations measured using the BCA assay. One milligram of whole-cell lysate was incubated with anti-TNIK antibody (Santa Cruz Biotechnology Inc., sc-136103) or its isotype control (Santa Cruz Biotechnology Inc., sc-3877) for 2 h at 4 °C. The lysates were then incubated with protein A/G-conjugated agarose (Santa Cruz Biotech., sc-2003) for 1 h at 4 °C, and centrifuged at 1000 × g for 5 min. to collect the precipitates. The precipitates were then washed four times with RIPA buffer and denatured using 20 µL of 1× SDS sample buffer. An identical concentration of whole-cell lysate was used as an input control. Western blot analysis using anti-TNIK (Cell Signaling Technologies, #32712) antibodies were performed to measure TNIK expression.
4
Immunoprecipitation and Western Blot Analysis
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Whole cell lysates (1 mg) were pre-incubated with non-immunized serum and protein A/G-conjugated agarose (Santa Cruz) for 1 hour at 4°C with a gentle rotation. After the centrifugation, the supernatants were further incubated with Gαh or PLC-δ1 (Gentex) antibodies and protein A/G-conjugated agarose overnight at 4°C with a gentle rotation. After several washes, the immunoprecipitates were resuspended in 20 μl of SDS-PAGE protein loading dye and boiled at 95°C. After the centrifugation, the supernatants were subjected to Western blot analysis.
Total protein (100 μg) from the designed experiments was separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were sequentially incubated with blocking buffer (5% nonfat milk in TBS containing 0.1% Tween-20) for 2 hours at room temperature, primary antibodies against Gαh, PLC-δ1 (Gentex), phosphorylated Akt, Akt, phosphorylated mTOR, mTOR, LC3-I/II and p62 (Cell Signaling) or GAPDH (AbFrontier) overnight at 4 °C, and peroxidase-labeled secondary antibodies for 1 hour at room temperature. At each step, the cells were extensively washed. Finally, immunoreactive bands were visualized by an enhanced chemiluminescence system (Amersham Bioscience).
Total protein (100 μg) from the designed experiments was separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were sequentially incubated with blocking buffer (5% nonfat milk in TBS containing 0.1% Tween-20) for 2 hours at room temperature, primary antibodies against Gαh, PLC-δ1 (Gentex), phosphorylated Akt, Akt, phosphorylated mTOR, mTOR, LC3-I/II and p62 (Cell Signaling) or GAPDH (AbFrontier) overnight at 4 °C, and peroxidase-labeled secondary antibodies for 1 hour at room temperature. At each step, the cells were extensively washed. Finally, immunoreactive bands were visualized by an enhanced chemiluminescence system (Amersham Bioscience).
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