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嗨~總蛋白你測(cè)了嗎？--關(guān)于蛋白磷酸化水平檢測(cè)兩三事
閱讀：1125 發(fā)布時(shí)間：2022-8-9
多種疾病的產(chǎn)生都伴隨著蛋白過(guò)度磷酸化的機(jī)制。研究磷酸化水平，以確定信號(hào)傳導(dǎo)。在不評(píng)估總蛋白磷酸化和非磷酸化水平的情況下，研究人員可能會(huì)得出錯(cuò)誤的結(jié)論，即一種化合物具有上調(diào)或下調(diào)磷酸化的作用，而實(shí)際上其作用可能是由于該蛋白質(zhì)表達(dá)水平的變化。這種錯(cuò)誤的分析結(jié)果導(dǎo)致對(duì)化合物的功能的判定。
HTRF®--均相時(shí)間分辨熒光，時(shí)間分辨（TRF）和熒光共振能量轉(zhuǎn)移（FRET）兩大核心技術(shù)的碰撞，利用兩個(gè)極其穩(wěn)定的熒光基團(tuán)，“相遇即發(fā)光"的原理，提供了一個(gè)簡(jiǎn)單（免洗），快速，高通量，高靈敏的方法分析磷酸化蛋白和總蛋白水平。
HTRF分析總蛋白和磷酸化蛋白
本研究進(jìn)行了總AKT和磷酸化AKT Ser473檢測(cè)，尤此提供總蛋白檢測(cè)重要性的依據(jù)。
實(shí)驗(yàn)方法：所有檢測(cè)方法均采用標(biāo)準(zhǔn)的貼壁細(xì)胞雙板HTRF檢測(cè)方法進(jìn)行。細(xì)胞與化合物在96孔板中孵育，裂解細(xì)胞，轉(zhuǎn)移到兩個(gè)不同的檢測(cè)板上，一個(gè)用于測(cè)量磷酸化AKT，另一個(gè)用于測(cè)量總AKT，并添加HTRF試劑進(jìn)行檢測(cè)。
HEK293細(xì)胞用于100000個(gè)細(xì)胞/孔的所有AKT檢測(cè)。細(xì)胞與不同濃度的IGF-1孵育10分鐘；已知的蛋白質(zhì)合成抑制劑環(huán)己胺，孵育16小時(shí)。數(shù)據(jù)均以HTRF Ratio值和歸一化值表示。
用IGF-1處理細(xì)胞以劑量依賴的方式顯著增加了AKT Ser473磷酸化水平，EC50值為1.06nM。IGF-1也下調(diào)了AKT的表達(dá)，半抑制濃度值為0.8nM。使用兩種分析數(shù)據(jù)計(jì)算的歸一化值P-AKT/T-AKT顯示，在IGF-1刺激下，磷酸化的AKT與非磷酸化的AKT的比例顯著增加。
環(huán)己酰亞胺處理AKT磷酸化水平降低，且AKT表達(dá)水平降低，且半抑制濃度值相似。如果沒(méi)有進(jìn)行總AKT檢測(cè)，并且只評(píng)估磷酸化AKT檢測(cè)結(jié)果，那么環(huán)己酰亞胺可能被錯(cuò)誤地定義為AKT磷酸化的抑制劑。通過(guò)進(jìn)行兩種分析和將數(shù)據(jù)表示為歸一化值，可以發(fā)現(xiàn)，AKT的磷酸化水平不受環(huán)己酰亞胺處理的調(diào)節(jié)。因此，環(huán)己酰亞胺可以被正確地識(shí)別為AKT表達(dá)的抑制劑，其半抑制濃度值為460nM，但它并不抑制磷酸化。
結(jié)合上述實(shí)驗(yàn)結(jié)論，可以幫助我們進(jìn)一步了解同時(shí)檢測(cè)磷酸化蛋白和總蛋白的重要性，正確并客觀的認(rèn)定化合物對(duì)蛋白磷酸化水平的調(diào)節(jié)。
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