T7-tagged purified recombinant HspB1 protein (1 μg) was added to 10 μg of EEF-tagged recombinant FLNCd18–21 in immunoprecipitation (IP) buffer [0.05% Triton X-100, 1% bovine serum albumin, protease inhibitors (Roche mini complete EDTA-free tablets) in phosphate-buffered saline (PBS)] and incubated for 1 hour, shaking at room temperature. This was followed by another 30-min incubation, shaking at room temperature after the addition of 0.5 μg of anti-T7 (69522, Novagen) or mouse IgG2b isotype (ab18457, Abcam) antibody. Next, 25 μl of protein G Sepharose resin (PC-G5, Generon) was added to each tube and incubated for 1 hour, shaking at room temperature. The beads were then washed four times with IP buffer, resuspended in 20 μl of 5× SDS sample buffer [312.5 mM tris-HCl (pH 6.8), 500 mM dithiothreitol (DTT), 10% SDS, 30% glycerol, 0.05% bromophenol blue], and boiled at 95°C for 5 min. Immunodetection was performed using the anti-T7 (69522, Novagen) or anti-EEF/anti-tubulin (YL1/2) (ab6160, Abcam) antibodies.
Ab18457
Manufactured by Abcam
Ab18457 is a lab equipment product manufactured by Abcam. It is designed for general laboratory use, but its core function is not specified in the available information. A more detailed and unbiased description cannot be provided while maintaining the requested approach.
Automatically generated - may contain errors
2 protocols using ab18457
1
Immunoprecipitation of HspB1 and FLNC
2
Flow Cytometry Analysis of ATP Synthase in MSCs
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MSCs (1 × 106) were seeded onto 10-cm culture dishes and incubated for 72 h. Cells were detached with 1 mM EDTA (J.T. Baker) and resuspended with MEM containing 20% FBS. After fixation, 1 × 106 MSCs were incubated with anti-ATP synthase complex immunocapture antibody [12F4AD8AF8] (ab109867, Abcam) (58 (link)), which is able to detect the native folded whole enzyme, or mouse anti-IgG antibody (ab18457, Abcam) overnight. The cells were then hybridized with Alexa Fluor 488–conjugated goat anti-mouse IgG secondary antibody (Abcam) by rotation for 2 h at room temperature. Triton X-100 (0.1%) in PBS was used for permeabilization. The fluorescence signal was detected using a BD FACSCanto II (BD Biosciences).
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