Glyoxyl agarose has been frequently used for multipoint covalent attachment of protein (including antibody and enzyme) and is particularly suited for enzyme immobilization when the stability of the immobilized enzyme is a concern (Selected review ref.: Zucca P. et. al. Molecules 2016, 21, 1577).
CellMosaic? designed this kit to work directly with glyoxyl agarose. Glyoxyl groups are aliphatic aldehyde groups with low steric hindrance towards the immobilization reaction and high stability at alkaline pH. Glyoxyl reacts with the amino group, forming Schiff’s base (imino bond). The imino bond is not very stable and can be reversible, so only proteins that form several imino bonds will be able to stay. For this reason, glyoxyl agarose permits the immobilization of the protein through the area with the highest density of lysine groups, where the highest likelihood of multipoint covalent attachment may be achieved. This multiple covalent attachment also allows appropriate alignment of the protein on the surface. After immobilization, the double bond of the Schiff’s base is finally reduced to form a stable secondary amino bond. The remaining aldehyde groups are converted into inert hydroxyl groups.
Proteins such as antibody containing disulfide bridges or enzyme bearing a metal ion in the active center may be affected by the final reducing step and should be used cautiously with this method.
產品描述
乙醛酰瓊脂糖經常用于蛋白質（包括抗體和酶）的多點共價連接，并且當關注固定化酶的穩定性時，特別適合于酶固定化（選定綜述參考文獻：Zucca P. et.等人，Molecules 2016，21，1577）JellMosaic ?設計該試劑盒以直接與乙醛氧基瓊脂糖一起工作。乙二氧基是脂肪族醛基，對固定化反應具有低空間位阻，在堿性pH下具有高穩定性。乙二氧基與氨基反應，形成席夫堿（亞氨基鍵）。亞氨基鍵不是很穩定，并且可以是可逆的，因此只有形成幾個亞氨基鍵的蛋白質才能夠留下來。由于這個原因，乙醛酰瓊脂糖允許通過具有最高密度的賴氨酸基團的區域固定蛋白質，其中可以實現多點共價連接的最高可能性。這種多重共價連接還允許蛋白質在表面上的適當對齊。固定化后，席夫堿的雙鍵最終被還原形成穩定的仲氨基鍵。剩余的醛基轉化為惰性羥基。蛋白質如含有二硫鍵的抗體或活性中心含有金屬離子的酶可能會受到最終還原步驟的影響，因此應謹慎使用該方法。CellMosaic?設計該試劑盒直接與戊二醛與氨基瓊脂糖反應衍生的戊二醛瓊脂糖（約20 μmol氨基/mL樹脂）一起使用。在這種情況下，戊二醛的一個醛基連接到瓊脂糖的氨基上，并留下其他醛在非常溫和的條件下與蛋白質的氨基反應。由于共價鍵和與固體載體中的殘余氨基的相鄰離子相互作用，最終產物相對穩定。比如說，用蛋白A對該試劑盒進行的初始測試提供了每mL沉降珠3 mg蛋白A的最大負載能力和每mL沉降珠8 mg人IgG的結合能力。（包括抗體和酶），并已用于蛋白質固定通過共價連接到氨基活化的基質或通過單純的交叉，蛋白質-蛋白質聚集體或吸附在氨基活化基質上的蛋白質的連接（精選綜述：巴博薩，O. RSC Avd. 2014，4，1583-1600; Fernando Lopez-gallego等人，Immobilization of Enzymes and cells：third edition，Methods in Molecular Biology，2013，vol.1051，p33-41中的第3章）。戊二醛瓊脂糖常用于溫和反應條件下的蛋白質固定，特別適用于不穩定蛋白質。
