Part:BBa_K3117026:Experience

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Applications of BBa_K3117026

The construct 2a (K2a) was synthesized by IDT. Accuracy of the inserted DNA after cloning was confirmed by sequencing. The data is depicted in Fig. 1.

Bild sequencing K2a Figure 1: Sequencing data of K2a

Fig.2 shows the western blot of the harvest after transfection into HEK 293T cells. For detection, the His-Tag in the K2a sequence (BBa_K3117005) provides the opportunity to be used as a target for a primary antibody in a western blot. K2a can be seen at expected height (42 kDa). Blurry bands might resulted from protease degradation. The presence of K2a in the medium proves the function of the Igk leader (BBa_K3117006), which is directing the protein into the secretory pathway.

Bild Ernte Figure 2: Western blot of the harvest after transfection into HEK 293T cells with an anti-His-Tag antibody

Furthermore the His-Tag (BBa_K3117005) in K2a allows purification of the protein with HisTrap columns. The western blot after purification is shown in Fig. 3, depicting the remaining presence of the protein after this step.

Bild Aufreinigung Figure 3: Western blot of the purified protein with HisTrap columns with an anti-His-Tag antibody

For the SpyTag/SpyCatcher reaction the protein K2a was added to the protein K2b (BBa_K3117030). A height of 83 kDa was expected in the afterwards performed western blot, depicted in Fig 4. A specific band appeared after adding the two products, but this band was running at ~120 kDa. This size might be explained by formation of trimeric protein (Schoene, Fierer, Bennett, & Howarth, 2014).

Bild Tag/Catcher Figure 4: Western blot after SpyTag/SpyCatcher reaction

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