Proteína Biotinilada

Serie PrecisionAvi

Los productos de esta serie se fabrican exclusivamente con tecnología Avitag™. Brevemente, un péptido único de 15 aminoácidos, la etiqueta Avi, se introduce en la proteína recombinante durante la construcción del vector de expresión. El único residuo de lisina en la etiqueta Avi es biotinilado enzimáticamente por la biotina ligasa BirA de E. Coli.

Esta técnica de marcaje enzimático de punto único ofrece muchas ventajas para los ensayos de unión de uso común.

• La biotinilación solo ocurre en el residuo de lisina de la etiqueta Avi.

• NO hay interferencia con las actividades de unión natural de la proteína objetivo.

• La orientación de la proteína es uniforme cuando se inmoviliza sobre una superficie recubierta de avidina.

Todos los productos

Serie UltraLys

Los productos de esta serie se fabrican utilizando nuestro enfoque de etiquetado químico desarrollado internamente. Las aminas primarias en las cadenas laterales de los residuos de lisina y el extremo N de la proteína se conjugan con biotinas, lo que generalmente da como resultado la unión de múltiples biotinas en una sola molécula de proteína.

• Mayor sensibilidad de detección

• Enfoque de etiquetado químico desarrollado internamente

Todos los productos

Lo más cercano a la conformación y modificación natural

La producción de nuestras proteínas recombinantes, incluidas las proteínas biotiniladas, se lleva a cabo utilizando nuestra plataforma de expresión patentada HEKMax®. Como anfitriones de expresión, las células HEK293 humanas tienen una variedad de ventajas en comparación con otros tipos de células, como se resume en la siguiente tabla.

Alta bioactividad y sensibilidad de detección

La bioactividad de las proteínas biotiniladas está determinada tanto por la estructura de la propia proteína como por la forma en que se realiza la biotinilación. Para cada proteína individual, probamos múltiples opciones de etiquetas y métodos de biotinilación, y evaluamos los productos en una variedad de ensayos de unión. Solo aquellos con el mejor rendimiento son seleccionados para la producción. La Figura 1 es un ejemplo de nuestros experimentos de evaluación interna:

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.

                 

Baja variación de lote a lote

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.