Frozen kidney sections were blocked with 5% bovine serum albumin for 30 min at 37 °C and the cell-climbing film (cells growing on a glass slide) was fixed in 4% paraformaldehyde for 30 min at 4 °C. The sections were incubated with a mixture of primary antibodies (nephrin guinea pig polyclonal antibody, 1:50, Progen Biotechnik; c-Abl rabbit polyclonal antibody, 1:50, Cell Signaling Technology) overnight at 4 °C. Fluorescein isothiocyanate (FITC)/tetramethylrhodamine-conjugated IgG was used as secondary antibodies at 37 °C for 90 min in the dark. All microscopic images were recorded using a confocal microscope (Olympus, Japan).
C abl rabbit polyclonal antibody
Manufactured by Cell Signaling Technology
The C-Abl rabbit polyclonal antibody is a laboratory reagent used for the detection and analysis of the Abl (Abelson murine leukemia viral oncogene homolog) protein in biological samples. The antibody recognizes the C-terminal region of the Abl protein.
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Lab products found in correlation
3 protocols using c abl rabbit polyclonal antibody
1
Immunofluorescence Staining of Kidney Sections
2
Co-Immunoprecipitation for Protein-Protein Interactions
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Co-immunoprecipitation experiments were performed using a kit (Beyotime, China) according to the manufacturer’s instructions. Briefly, total protein from cultured cells was extracted using lysis buffer (20 mM Tris, 150 mM NaCl, 1.0% Triton X-100, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5). A c-Abl rabbit polyclonal antibody (1:200, Cell Signaling Technology) or myc mouse monoclonal antibody (5 μg/500 μg total protein, Medical and Biological Laboratories) was added to the protein samples, which were then rotated overnight at 4 °C. Then, the samples were mixed with 40 µl of protein A+G agarose beads and incubated for 3 h at 4 °C. The beads were mixed with 1 × Lane Marker Sample Buffer. After being boiled at 95–100 °C for 5 min, the samples were analyzed by western blotting.
3
Protein Interactions: c-Abl, Nephrin, and SHIP2
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Coimmunoprecipitation experiments were performed according to the manufacturer’s instructions (P2012; Beyotime, China). The total proteins from the cultured podocytes were extracted using lysis buffer (20 mM Tris, 150 mM NaCl, 1.0% Triton X-100, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5). A c-Abl rabbit polyclonal antibody (1:200; Cell Signaling Technology) was added to the protein samples, and these were then rotated overnight at 4°C. The mixture was then loaded with 40 μl of protein A+G–agarose, incubated for 3 h at 4°C, centrifuged at 2500 × g and 4°C for 5 min and washed five times with phosphate-buffered saline (PBS). The beads were mixed with 1× Lane Marker Sample Buffer. After being boiled at 95–100°C for 5 min, the samples were analyzed by Western blotting for c-Abl, nephrin, and SHIP2 expression.
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