蛋白酶活性檢測試劑盒 | Protease Activity Assay Kit 貨號701410 

Cayman 的蛋白酶活性檢測試劑盒(Cayman’s Protease Activity Assay Kit,  貨號701410)提供了一種方便的方法確定樣品中蛋白酶的活性。蛋白水解消化FITC-酪蛋白底物(FITC-casein substrate)可以通過螢光的變化來監測偏振 (fluorescence polarization, FP) 或總螢光。螢光是分子的能力吸收入射（激發）光子的能量，然後重新發射作為一種新的、能量稍低的（emission）光子的能量。一個小螢光分子將在非常小的時間間隔內明顯旋轉光子的吸收和螢光光子的發射。如果激發光是偏振的(polarized)，這種旋轉將導致平面的隨機化發出的光。因此，小螢光分子使激發脈衝去極化的偏振光。像 FITC-酪蛋白(FITC-casein)這樣的大螢光分子不會旋轉明顯在相同的小時間間隔內。因此，它們將發出光保留了偏振激發光的一些偏振。這種極化被量化為毫極化單位，或 mP。在此測定中，總 FP 減少因為 FITC-酪蛋白(FITC-casein)被消化成更小、旋轉更快的螢光素標記碎片。如果無法測量 FP，也可以通過總螢光的變化測量以確定蛋白酶活性。 FITC 與酪蛋白的結合結果適度猝滅總螢光。這種螢光隨著FITC-酪蛋白(FITC-casein)底物被消化成較小的螢光素標記片段。兩種測定形式均使用 480-495 nm 的激發波長和發射波長波長為 515-525 nm。

 

 

Cayman’s Protease Activity Assay Kit provides a convenient method for determining the activity of proteases in samples. Proteolytic digestion of the FITC-casein substrate can be monitored by changes in either fluorescence polarization (FP) or total fluorescence.¹ Fluorescence is the ability of a molecule to absorb the energy of an incoming (excitation) photon and then re-emit this energy as a new, slightly less energetic (emission) photon. A small fluorescent molecule will rotate appreciably during the very small interval of time between absorption of a photon and emission of the fluorescence photon. If the excitation light is polarized, this rotation will result in randomization of the plane of the emitted light. Thus, small fluorescent molecules depolarize an excitation pulse of polarized light. Large fluorescent molecules, like FITC-casein, do not rotate as appreciably in the same small interval of time. They will, therefore, emit light that retains some of the polarization of the polarized excitation light. This polarization is quantified as millipolarization units, or mP. In this assay, the total FP decreases as the FITC-casein is digested into smaller, quicker rotating fluorescein-labeled fragments. If measuring FP is not available, the change in total fluorescence can also be measured to determine protease activity. The conjugation of FITC to casein results in moderate quenching of the total fluorescence. This fluorescence increases as the FITC-casein substrate is digested into smaller fluorescein-labeled fragments. Both assay formats use an excitation wavelength of 480-495 nm and an emission wavelength of 515-525 nm.