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As a crucial part of the selection system, the correct expression of the binding and the target protein is inevitable. By testing several protein samples via SDS PAGE and western blot, this could be verified.
Figure 1. SDS PAGE for expression control: Result of a SDS PAGE with samples from left to right: Color prestained marker, HA4 Evobody wild type, HA4 Y87A mutant Evobody, HA4 R38A mutant Evobody, HA4 R38A E52A, SH2:cI + HA4 Evobody, SH2:cI434, SH2:cILambda
As the estimated molecular weight for the HA4 Evobody is 22 kDa and for the SH2 fusion protein 24 kDa, bands were expected to occur in the lower half of the separation gel.
It can be seen in Figure 1 that the samples that should contain the HA4 Evobody and the SH2:cI fusion protein together, the SH2:cI434 fusion protein alone and the SH2:cILambda fusion protein alone show a band visible at an approximate molecular weight of 24 kDa. As the bands are relatively strong in comparison to the native proteins, which form the other bands in the gel, it can be assumed that the SH2 fusion proteins were expressed correctly. However, the bands which can be seen in all the samples that should contain the HA4 Evobody or one of its mutants(BBa_K2082222, BBa_K2082223 and BBa_K2082224) alone show no extraordinary expression, which lead to the assumtion that HA4 might not be expressed in the samples. To further prove that the HA4 Evobody was, after all, expressed, a western blot was done afterwards.
Western Blot
By blotting a previosly prepared SDS gel similar to the one explained before but missing the SH2 samples, a western blot was done with an anti-myc antibody as the primary antibody and an anti-mouse antibody with a horsereddish peroxidase fused to it as the secondary antibody. The primary antibody was chosen because the HA4 Evobody and its mutants contain a myc-tag as a linker between the HA4 binding protein and the RpoZ. The secondary antibody has been chosen because the primary antibody was a mouse derived antibody.
Figure 2. Western blot for expression control: Result of a western blot with samples from left to right: marker, HA4 Evobody wild type, HA4 Y87A mutant Evobody, HA4 R38A mutant Evobody, HA4 R38A E52A, SH2:cI+HA4 Evobody
It can be seen in Figure 2 that all samples blotted show a band at an approximate weight of 22 kDa. As the primary antibody used in the western blot should only bind to a myc-tag, it can be assumed that the bands visible verify the correct expression of the HA4 Evobody and its mutants in the samples.
BLItz
To confirm that the anti-myc antibody binds correctly to the HA4 Evobody, a bio-layer interferometry was performed with a BLItz system by Forté Bio.
Figure 3. BLItz results of immobilized HA4 with anti-myc antibody: Result of a bio-layer interferometry of HA4 with anti-myc antibody on Amine reactive 2nd generation biosensor.
The HA4 Evobody was immobilized on an amine reactive 2nd generation biosensor by Forté Bio. As the secondary supplement, the anti-myc antibody previously used in the western blot was used. As it can be seen in Figure 3, after the addition of the anti-myc antibody a wavelength shift occured, which shows that an association of the anti-myc antibody to the HA4 Evobody happened. When a regeneration solution was added, a dissociation of the anti-myc antibody could be observed.
With this results put together, it can be confirmed that both the HA4 Evobody and its mutants as well as both SH2:cI fusion proteins were correctly expressed.
