ビオチン標識タンパク質

PrecisionAviシリーズ

このシリーズのビオチン標識製品はAviTag™ 技術を用いて製造されております。15アミノ酸ペプチドであるAviタグは発現ベクター構築の際にリコンビナントタンパク質に組み込まれ、Aviタグの単一のリジン残基はE. ColiオチンリガーゼBirAによって酵素的なビオチン化をされます。

このシングルポイント酵素標識技術は通常の結合アッセイに適していると考えられています。

• ビオチン化はAviタグのリジン残基にのみ施されます。

• タンパク質の元の結合部位は干渉されません。

• アビジンコートプレートへ固定する際には配向を均一に保つことが可能です。

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UltraLysシリーズ

このシリーズの商品は弊社独自の化学的修飾法を用いて製造されています。リシン残基側鎖のアミンおよびタンパク質N末端の第一級アミンがビオチン化されます。通常、一つのタンパク質分子に対し複数のビオチンが結合しています。

• 高い検出感度

• 弊社独自の化学的修飾法を使用されている。

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天然構造のコンフォメーションと修飾に最も近い

ビオチン化タンパク質を含む弊社のタンパク質は、独自技術のHEKMax®発現プラットフォームを用いて製造しています。ヒトHEK293細胞にはタンパク質発現宿主として、他の細胞と比較し多くの優れた点があります。

高い生物活性＆検出感度

ビオチンタンパク質の生物活性は、タンパク質自体の構造と、ビオチン化の方法により決定されます。我々はすべてのタンパク質に対して、様々なタグの種類とビオチン化方法を検証し、様々な結合実験により評価をしています。最も良い組み合わせで製品が開発されます。

Fig. 1 Binding activity of different forms of biotinylated PD1 evaluated in a functional ELISA against rhPD-L1 (Cat. No. PD1-H5258).

Our biotinylated proteins demonstrate high bioactivity and superb detection sensitivity in different application. In functional ELISA, the biotinylated human TACI has high binding activity with BAFF as showed in Fig. 2. Interestingly, oriented immobilization of biotinylated CD155 offers much higher sensitivity than random immobilization of unconjugated CD155 as showed in Fig. 3. Fig.4 and Fig. 5 show that the biotinylated PD1 can be well applied to AlphaLISA to detect the binding activity with its ligand PD-L1 and PD-L2. The Fig. 6 and Fig. 7 show that the biotinylated protein can be easily used for determining the affinity between protein and therapeutic antibody in SPR assay. Also, the biotinylated proteins are often used in cell based assay, such as cytotoxicity assay and evaluation of CAR expression (Fig. 8 & Fig. 9).

Fig.2 Immobilized Human BAFF, Fc Tag, active trimer (Cat. No. BAF-H5261) at 1 μg/mL (100 μL/well) can bind Biotinylated Human TACI, Fc,Avitag (Cat. No. TAI-H82F6) with a linear range of 0.002-0.05 μg/mL.

Fig. 3 Comparison of oriented immobilization (biotinylated CD155 on precoated streptavidin plate, Cat. No. CD5-H82E3) and random immobilization (unconjugated CD155 direct coating on 96-well plate, Cat. No. CD5-H5223）binding to TIGIT（ Cat. No. TIT-H5254）. Oriented immobilization of biotinylated CD155 offers much higher sensitivity than random immobilization of unconjugated CD155.

Fig 4. Biotinylated Human PD-1（Cat. No. PD1-H82F1）at 1 μg/mL (5 μL/well) can bind Human PDL1 (Cat. No. PD1-H5229) with a linear range of 0.02-0.625 μg/mL.

Fig 5. Biotinylated Human PD-1（Cat. No. PD1-H82F1）at 1 μg/mL (5 μL/well) can bind Human  PDL2 (Cat. No. PD2-H5220) with a linear range of 0.02-0.625 μg/mL.

Fig. 6 Immobilized biotinylated human VEGF165 (Cat. No. VE5-H82Q0) on CM5 Chip via streptavidin, can bind Avastin with an affinity constant of 0.417nM as determined in SPR assay (Biacore T200).

Fig.7 Loaded Biotinylated Human PD-1, Avitag,His Tag (recommended for biopanning) (Cat. No. PD1-H82E4) on SA Biosensor, can bind Human PD-L1, His Tag (HPLC verified) (Cat. No. PD1-H5229) with an affinity constant of 2.4 μM as determined in BLI assay (ForteBio Octet Red96e).

Fig. 8 Recombinant human TNF-alpha (Cat. No. TNA-H82E3) induces cytotoxicity effect on the WEH1-13VAR cells in the presence of the metabolic inhibitor actinomycin D. The EC50 for this effect is 0.014-0.029 ng/ml. The result shows that the biotinylated human TNF-alpha is consistent with naked TNF-alpha in cytotoxicity assay.

                 

ロット間のばらつきが小さい

We routinely apply rigorous quality control measures to ensure consistent performance of our products. Newly produced products are subjected to side-by-side comparison with our internal standard in a variety of assays. Only those within an acceptable margin of difference are allowed to be released. As demonstrated in Fig. 9, three different batches of biotinylated hTNF-alpha (Cat. No. TNA-H82E3) are tested and compared using a standard ELISA analysis against Adalim*mab, the result shows that the batch variation among the tested samples is negligible. The batch-to-batch conformance is also good as displayed in cell based assay (Fig. 10).

Fig. 10 In the above ELISA analysis, three different lots of biotinylated hTNF-alpha (Cat. No. TNA-H82E3) were used detect immobilized Adalim*mab (0.5 ug/ml). The result showed that the batch variation among the tested samples is negligible.

Fig. 11 Recombinant biotinylated human TNF-alpha (Cat. No. TNA-H82E3) induces cytotoxicity effect on the WEH1-13VAR cells in the presence of the metabolic inhibitor actinomycin D. The EC50 for this effect is 0.029-0.052 ng/ml.