Western blot cells were harvested and then lysed on ice using cell lysis buffer (Beyotime, China) with 1% protease inhibitor Cocktail Set III (Merck, Germany). The collection of protein fractions was done by 5 min centrifugation at 15,000 g, 4°C and then subjected to SDS-PAGE. Afterward, the protein fractions were transferred onto an NC membrane (PALL, USA). After treatment with 5% skim milk, the membrane was incubated with specific antibodies at 4°C overnight. After that, a secondary antibody was added labeled with HRP (CoWin Bioscience, Beijing, China). Visualization was performed by using a chemiluminescence detection system (Progma, USA) as recommended by the manufacturer. The primary antibodies that were used included MACF1 rabbit pAb (1:500, Abcam, USA), β-catenin rabbit pAb (1:1,000, Cell Signaling Technology, USA), and GAPDH mouse mAb (1:1,000, Calbiochem, USA). All experiments were repeated three times.
Macf1 rabbit pab
Manufactured by Abcam
Sourced in United States
MACF1 rabbit pAb is an antibody that targets the microtubule-actin crosslinking factor 1 protein. It is a polyclonal antibody produced in rabbits.
Automatically generated - may contain errors
2 protocols using macf1 rabbit pab
1
Western Blot Analysis of Cell Signaling
2
Osteoblast Protein Extraction and Western Blot
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The osteoblasts were harvested and then lysed with Cell Lysis Buffer (Beyotime, China) containing a protease inhibitor cocktail (Merck, Germany) on ice. The protein extracts were collected after 4 °C centrifugation (15,000 g for 5 min) and then boiled with SDS-PAGE protein loading buffer (Biosharp, China). The protein was transferred to nitrocellulose membranes (PALL, USA) after separation via 8% SDS-PAGE (MACF1) or 10% SDS-PAGE (COLIα1 and GAPDH). The separated protein fractions on the membranes were blocked with 5% skim milk and incubated overnight at 4 °C with specific primary antibodies on a shaking device. HRP-labeled secondary antibody (Beijing CoWin Bioscience, China) was added after primary antibodies rinsed off, and then protein bands were visualized using ECL chemiluminescence reagents (Rockford, United States) by X-ray film (Kodak, NY) or T5200 Multi Chemiluminescence Detection System (Tanon, China). The primary antibodies included MACF1 Rabbit pAb (1:1000, Abcam, USA), COLIα1 Rabbit pAb (1:1000, Cell Signaling Technology, USA) and GAPDH Rabbit pAb (1:1000, Calbiochem, Germany) 23 (link).
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