Brain tissues or cultured cells were harvested in a lysis buffer (Beyotime, Cat# P0013, China) containing a protease and phosphatase inhibitor cocktail (Beyotime, Cat# 1045, China) at 4 °C for 4 h, and centrifuged at 4 °C (12 000 rpm) for 10 min. According to the manufacturer's instructions, Co-IP experiments were performed using the Pierce™ Crosslink Magnetic Co-IP Kit (Thermo Fisher Scientific, Cat# 26149, USA). Magnetic beads were crosslinked with non-specific rabbit (1:50, Abclonal, Cat# AC005, China) or mouse IgG (1:50, Abclonal, Cat# AC011, China), rabbit anti-Shank3 (1:50, Cell Signaling Technology, Cat# 64555, USA), mouse anti-STIM1 (1:50, Thermo Fisher Scientific, Cat# MA1-19451, USA), rabbit anti-HA (1:100, Proteintech Group, Cat# 51064-2-AP, USA). The beads were then washed three times with coupling buffer. The protein extracts mixed with the beads were incubated overnight at 4 °C. Following the magnetic isolation, the precipitates were eluted with elution buffer, neutralized with neutralization buffer, and prepared for Western blot.
Rabbit anti shank3
Manufactured by Cell Signaling Technology
Sourced in China, United States
Rabbit anti-Shank3 is a primary antibody that specifically recognizes the Shank3 protein. Shank3 is a scaffold protein that plays a crucial role in the organization and function of the postsynaptic density in neuronal cells. This antibody can be used for the detection and analysis of Shank3 in various applications, such as Western blotting and immunohistochemistry.
Automatically generated - may contain errors
2 protocols using rabbit anti shank3
1
Co-Immunoprecipitation of Protein Complexes
2
Automated Capillary Western Blotting Analysis
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Capillary western blot analyses were carried out using the chemiluminescent and fluorescent western blotting Jess system with a 12–230 kDa Jess separation module (ProteinSimple, Santa Clara, CA, USA) in accordance with the manufacturer's instructions. Protein concentration was measured using the BCA method (Vazyme, Nanjing, Jiangsu, CN). Protein samples were mixed with 0.1× sample buffer and 5× master mix (ProteinSimple) to achieve a final protein concentration of 1 µg µL−1 and then denatured at 95 °C for 5 min. Then, the denatured protein samples, blocking buffer, primary antibodies, HRP‐conjugated secondary antibodies, wash buffer, and chemiluminescent substrate (1:1 luminol‐peroxidase mixture) were added to specific wells of the assay plate. Rabbit anti‐YBX1 (1:10; Cell Signaling Technology, Boston, MA, USA), rabbit anti‐SHANK3 (1:10; Cell Signaling Technology), and rabbit anti‐GAPDH (1:50; HuaBio, Hangzhou, Zhejiang, CN) antibodies were used as primary antibodies. Anti‐rabbit antibodies (ProteinSimple) were used as secondary antibodies. After loading the plate, separation electrophoresis was performed in the capillary system and immunodetection was fully automated. The relative expression of proteins was automatically calculated using Compass for Simple Western software (version 5.0; ProteinSimple). All experiments were repeated three times.
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