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AssayGenie高靈敏度脂肪酸氧化 (FAO) 檢測試劑盒的應(yīng)用

更新時(shí)間：2024-01-18 點(diǎn)擊次數(shù)：2616

脂肪酸每碳提供的ATP比葡萄糖等碳水化合物多。在具有高能量需求的組織中，例如心臟組織，高達(dá) 50 - 70%的能量來自脂肪酸β-氧化，可轉(zhuǎn)化脂肪酸制乙酰輔酶A，同時(shí)產(chǎn)生FADH2和NADH 線粒體。

這種嚴(yán)格的有氧氧化途徑由四個(gè)不同的步驟組成：酰基輔酶A脫氫酶脫氫，烯酰輔酶A水合水合酶，通過 3-羥基酰輔酶 A 脫氫酶和硫解酶脫氫 β-酮硫醇酶裂解。

研究脂肪酸氧化的重要性

β-氧化系統(tǒng)在人體中的重要性體現(xiàn)在存在一組被歸類為脂肪酸氧化的遺傳疾病疾病（FAOD），這是一組異質(zhì)性脂肪酸缺陷轉(zhuǎn)運(yùn)和氧化，并遺傳為常染色體隱性遺傳病，具有廣泛的臨床表現(xiàn)。

此外，有證據(jù)表明，糧農(nóng)組織在糧農(nóng)組織活動(dòng)期間的監(jiān)管胎兒發(fā)育不僅對胎兒生命很重要，而且對成年期健康和疾病的影響。糧農(nóng)組織的衡量標(biāo)準(zhǔn) 因此，活動(dòng)為病理生理學(xué)和病理學(xué)提供了有價(jià)值的見解許多疾病的代謝基礎(chǔ)。

檢測精靈非放射性糧農(nóng)組織的測定基于底物辛酰輔酶A的氧化。生成 NADH與四唑鹽INT還原為甲胺素偶聯(lián)。紅色甲臜的強(qiáng)度與糧農(nóng)組織的增加成正比活動(dòng)。測定溶液和底物應(yīng)等分儲存在 -80°C.

Assay Genie檢測精靈脂肪酸氧化檢測試劑盒是一種高度靈敏的試劑盒用于定量測量脂肪酸的比色法細(xì)胞和組織中的氧化。

這種檢測方法利用高可溶性辛酰輔酶A底物，可實(shí)現(xiàn) 脂肪酸氧化與長鏈低溶性的測量底物如18C不飽和脂肪酸油酸酯。

上圖：脂肪酸氧化測定：組織裂解物由健康的倉鼠心臟和與心力衰竭相關(guān)的TO2倉鼠模型。裂解物用于測定和脂肪酸氧化水平量化。

下圖：脂肪酸氧化測定：辛酰輔酶A是底物，而不是反應(yīng)的產(chǎn)物。請見圖。

細(xì)胞/組織提取物的制備：

1.用冰冷的磷酸鹽緩沖鹽水（PBS）洗滌~106細(xì)胞兩次，并從細(xì)胞沉淀中去除PBS。細(xì)胞沉淀應(yīng) 儲存在-80oC.組織樣品應(yīng)用PBS洗滌至去除血細(xì)胞，這可能導(dǎo)致檢測結(jié)果不一致。

2.通過稀釋 1x 細(xì)胞裂解液制備足夠的 10x 細(xì)胞裂解液用冰冷的dH 2 O溶液。加入 50 – 100 μl 冰冷的 1x 細(xì)胞裂解細(xì)胞沉淀溶液。通過上下移液提取細(xì)胞（溫和）。將裂解物留在冰上5分鐘間歇性輕微激動(dòng)。如果裂解物是粘稠的，添加更多的1x細(xì)胞裂解溶液并重復(fù)移液。在以最大速度冷藏微量離心機(jī)5分鐘。回收上清液用于測定。將組織在 1x 細(xì)胞裂解液中勻漿，并裂解通過離心澄清。每 25.0 毫升 5x 使用 ~1 毫克組織用于組織勻漿的細(xì)胞裂解溶液。

3.進(jìn)行蛋白質(zhì)測定以確定樣品蛋白質(zhì)濃度。通過用冰冷的 1x 稀釋來標(biāo)準(zhǔn)化樣品蛋白質(zhì)濃度細(xì)胞裂解液至1 – 3毫克/毫升。將蛋白質(zhì)樣品保存在冰上次。凍融的粗蛋白裂解物可表現(xiàn)出酶還原活動(dòng)。裂解物應(yīng)儲存在-80oC。注意：不要使用緩沖液含有還原劑或SDS。

試劑解凍：

將解凍的糧農(nóng)組織化驗(yàn)溶液和20倍糧農(nóng)組織底物放在冰上。輕輕攪拌移液前的溶液。重要的是要盡量減少時(shí)間試劑解凍。使用后立即冷凍溶液。

對照溶液和反應(yīng)溶液的制備：

1.對照溶液由1份dH 2 O和20份混合制備糧農(nóng)組織化驗(yàn)解決方案，例如25μldH 2 O與500μlFAO化驗(yàn)混合溶液。測定過程中將新鮮制備的對照溶液保持在冰上。

2.反應(yīng)溶液通過混合1份20x FAO底物和 20份糧農(nóng)組織化驗(yàn)溶液，例如25μl 20x糧農(nóng)組織底物與 500μl糧農(nóng)組織測定溶液。將溶液放在冰上并立即使用。每個(gè)蛋白質(zhì)樣品用50μl對照溶液和50μl處理反應(yīng)溶液分為兩組。

Assay Genie檢測精靈脂肪酸氧化檢測試劑盒的應(yīng)用

研究健康和病理狀態(tài)（脂質(zhì)代謝）的代謝途徑和過程。

信號轉(zhuǎn)導(dǎo)途徑的研究。

遺傳性疾病的研究，例如脂肪酸氧化障礙（FAODs）*。

心血管疾病的研究，例如心力衰竭。

胎兒發(fā)育過程中細(xì)胞能量代謝的研究。

脂肪酸氧化在癌癥代謝中的影響。

*此試劑盒僅供研究使用，不能用于診斷人類疾病。

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PACO06340-50	CDK2 Antibody (PACO06340)	50ug	assaygenie
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