Western blot

Proteins (30μg) extracted from cells were loaded and separated in 8% SDS-PAGE and then transferred on nitrocellulose membranes. The membranes were blocked with 5% non-fat milk and incubated with Anti-SP1 (1:1000, 9389, Cell Signaling Technology, MA, USA) or Anti-CCR7 (1:1000, ab32527, Abcam, Cambridge, UK) in 5% milk/PBS buffer at 4℃ overnight, followed by incubation with goat anti-rabbit secondary antibody (A23720, Abbkine, Wuhan, China) conjugated with DyLight fluorescent 680. After extensive wash with PBST buffer, the band of protein was visualized under Odyssey CLx imaging system (LI-COR, Inc., NE, USA).从细胞中提取的蛋白质（30μg）经上样后，于8%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中完成分离，随后转印至硝酸纤维素膜。以5%脱脂牛奶对膜进行封闭，接着在4℃条件下，用含5%脱脂牛奶的磷酸盐缓冲盐水（PBS）缓冲液配制的抗SP1（Anti-SP1）一抗（稀释比例1:1000，货号9389，美国马萨诸塞州Cell Signaling Technology公司）或抗CCR7（Anti-CCR7）一抗（稀释比例1:1000，货号ab32527，英国剑桥Abcam公司）孵育过夜。随后，使用偶联DyLight荧光染料680的山羊抗兔二抗（货号A23720，中国武汉Abbkine公司）进行孵育。经磷酸盐缓冲盐水-吐温20（PBST）缓冲液充分洗涤后，借助Odyssey CLx成像系统（美国内布拉斯加州LI-COR公司）实现蛋白条带的可视化检测。

搜集汇总

背景与挑战

背景概述

该数据集记录了Western blot实验的详细流程，包括蛋白质提取、电泳分离、抗体孵育和成像分析等步骤，特别关注头颈鳞状细胞癌相关蛋白SP1和CCR7的检测。数据集采用CC BY 4.0许可协议，主要用于癌症和细胞生物学领域的研究。