Cells in the exponential growth phase were collected and washed. Ice-cold lysis buffer was added, and the cell suspension was sonicated. The supernatant was collected and the total protein concentration was determined. Samples (500 μg) were then incubated with 2 μg of YWHAE (1:100, sc-23957, Santa Cruz Biotechnology, Santa Cruz, CA) or HE4 (1:1500, ab200828, Abcam, Cambridge, UK) primary antibodies. An IgG antibody (5145S, Cell Signaling Technology Inc., Danvers, MA) of the same species as the primary antibodies was used as a negative control. Following mixing, the samples were rotated slowly overnight at 4 °C. Subsequently, Protein A/G Plus-Agarose Beads (sc-2003, Santa Cruz Biotechnology, Santa Cruz, CA) were added to each tube and incubated for 6 h. Bound proteins were collected via centrifugation, (2×) loading buffer was added, and the samples were boiled to denature the proteins.
Igg antibody 5145s
Manufactured by Cell Signaling Technology
IgG antibody (5145S) is a laboratory reagent used in various research applications. It is a purified immunoglobulin G (IgG) antibody that can be used for detecting and quantifying target proteins in biological samples.
Automatically generated - may contain errors
2 protocols using igg antibody 5145s
1
Immunoprecipitation and Protein Extraction
2
Immunoprecipitation of YWHAE and HE4
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Cells in exponential growth were collected and washed. Iced lysis buffer was added and the cell suspension was sonicated. The supernatant was collected and total protein concentration was determined. Total protein samples (500 μg) were incubated with 2 μg of YWHAE (1 100, sc-23957, Santa Cruz Biotechnology, Santa Cruz, CA) or HE4 (1:1500, ab200828, Abcam, Cambridge, UK) primary antibody. An IgG antibody (5145S, Cell Signaling Technology, Beverly, MA) of the same species of the primary antibody was used as negative control. After mixing, the samples were left overnight at 4°C with slow rotation. Afterwards, beads (sc-2003, Santa Cruz Biotechnology, Santa Cruz, CA) were added to each tube and incubated for 6 h. The bound proteins were collected by centrifugation, (2×) Loading Buffer was added, and the samples were heated to denature the proteins.
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