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

更新時(shí)間：2024-01-18 點(diǎn)擊次數(shù)：2663

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(lái)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(guò) 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(duì)胎兒生命很重要，而且對(duì)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(jiàn)解許多疾病的代謝基礎(chǔ)。

檢測(cè)精靈非放射性糧農(nóng)組織的測(cè)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(cè)定溶液和底物應(yīng)等分儲(chǔ)存在 -80°C.

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

這種檢測(cè)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(zhǎng)鏈低溶性的測(cè)量底物如18C不飽和脂肪酸油酸酯。

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

下圖：脂肪酸氧化測(cè)定：辛酰輔酶A是底物，而不是反應(yīng)的產(chǎn)物。請(qǐng) 見(jiàn)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(chǔ)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(cè)結(jié)果不一致。

2.通過(guò)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(guò)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(cè)定。將組織在 1x 細(xì)胞裂解液中勻漿，并裂解通過(guò)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(cè)定以確定樣品蛋白質(zhì)濃度。通過(guò)用冰冷的 1x 稀釋來(lái)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(chǔ)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(duì)照溶液和反應(yīng)溶液的制備：

1.對(duì)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(cè)定過(guò)程中將新鮮制備的對(duì)照溶液保持在冰上。

2.反應(yīng)溶液通過(guò)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(cè)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(duì)照溶液和50μl處理反應(yīng)溶液分為兩組。

Assay Genie檢測(cè)精靈脂肪酸氧化檢測(cè)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(guò)程。

信號(hào)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(guò)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
BN00773-200	Nitrite Assay Kit (Griess Reagent) (BN00773)	200T	assaygenie
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CNDL00059	Canine Luteinizing Hormone (LH) ELISA Kit	96T	assaygenie
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RPPB3176	Human CHMP4A Recombinant Protein (RPPB3176)	5ug	assaygenie
RPPB4377	Human RAC1 Recombinant Protein (RPPB4377)	50ug	assaygenie
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