Detection of cellular fibronectin by In-Cell Western
AIM: To establish a method to detect cellular fibronectin(cFN) by In-Cell Western technique. METHODS: Osteoblast like cells (MG-63) cultured in microwells were fixed, blocked, and incubated with anti-FN monoclonal antibody. After being incubated with near-infrared fluorescence labeled secondary antibody, cells were scanned and analyzed by using Odysssey system. The method was further optimized with fixative, permeablization, blocking buffer and concentration of primary antibody. RESULTS: The cFN In-Cell Western method was validated for specificity by traditional western blot under optimized conditions and applied to detect cFN of osteoblast-like cell. The cFN signal of 4 × 103 cells per well can be detected and the standard curve is linear with MG-63 cell density when cell confluence is lower than 90% (approx. 1.6 × 104 cells per well). CONCLUSION: A rapid, high-throughput and cost-effective In-Cell Western method for detecting cFN was established and applied to extracellular matrix research successfully.
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万方数据
维普
