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多种疾病的产生都伴随着蛋白过度磷酸化的机制。研究磷酸化水平，以确定信号传导。在不评估总蛋白磷酸化和非磷酸化水平的情况下，研究人员可能会得出错误的结论，即一种化合物具有上调或下调磷酸化的作用，而实际上其作用可能是由于该蛋白质表达水平的变化。这种错误的分析结果导致对化合物的功能的判定。
HTRF®--均相时间分辨荧光，时间分辨（TRF）和荧光共振能量转移（FRET）两大核心技术的碰撞，利用两个极其稳定的荧光基团，“相遇即发光"的原理，提供了一个简单（免洗），快速，高通量，高灵敏的方法分析磷酸化蛋白和总蛋白水平。
HTRF分析总蛋白和磷酸化蛋白
本研究进行了总AKT和磷酸化AKT Ser473检测，尤此提供总蛋白检测重要性的依据。
实验方法：所有检测方法均采用标准的贴壁细胞双板HTRF检测方法进行。细胞与化合物在96孔板中孵育，裂解细胞，转移到两个不同的检测板上，一个用于测量磷酸化AKT，另一个用于测量总AKT，并添加HTRF试剂进行检测。
HEK293细胞用于100000个细胞/孔的所有AKT检测。细胞与不同浓度的IGF-1孵育10分钟；已知的蛋白质合成抑制剂环己胺，孵育16小时。数据均以HTRF Ratio值和归一化值表示。
用IGF-1处理细胞以剂量依赖的方式显著增加了AKT Ser473磷酸化水平，EC50值为1.06nM。IGF-1也下调了AKT的表达，半抑制浓度值为0.8nM。使用两种分析数据计算的归一化值P-AKT/T-AKT显示，在IGF-1刺激下，磷酸化的AKT与非磷酸化的AKT的比例显著增加。
环己酰亚胺处理AKT磷酸化水平降低，且AKT表达水平降低，且半抑制浓度值相似。如果没有进行总AKT检测，并且只评估磷酸化AKT检测结果，那么环己酰亚胺可能被错误地定义为AKT磷酸化的抑制剂。通过进行两种分析和将数据表示为归一化值，可以发现，AKT的磷酸化水平不受环己酰亚胺处理的调节。因此，环己酰亚胺可以被正确地识别为AKT表达的抑制剂，其半抑制浓度值为460nM，但它并不抑制磷酸化。
结合上述实验结论，可以帮助我们进一步了解同时检测磷酸化蛋白和总蛋白的重要性，正确并客观的认定化合物对蛋白磷酸化水平的调节。
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HTRF®需要使用搭载有相应模块的酶标仪进行读值，优宁维推荐PerkinElmer VICTOR Nivo™ 多功能酶标仪：
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