December 11, 2019
by Faiz Kahn
Edited by Elizabeth Jergens
Recap from previous month’s update:
1) We had successful conjugation of DNA to the QDs and to AuNPs for photoswitching experiments
2) A good blocking buffer was found for cell labelling using DNA cages, but some issues with DNA conjugation to antibodies were still being seen.
Figure 1. Comparison of antibodies conjugated to DNA and unconjugated antibodies that underwent the same treatment.
U87 cells were stained with DAPI (blue) for the nucleus, primary antibodies targeting beta-1 integrin, and secondary antibodies with a red fluorophore. The positive control was stained with pristine secondary antibodies. The blank antibodies are a sample that was treated the same as the experimental antibody without the addition of the linker used in conjugation, so they don’t have any DNA attached to them. The experimental conditions had antibodies that were conjugated to the DNA and had cages attached to them. Experiment 1 was run at an antibody concentration of 1:200 and experiment 2 was 1:500. Based on these results, we believe that during the step of adding the linker to the antibody we are killing the antibodies. The next step trying another that we have and then switching to a copper-free click chemistry procedure.
Recently, in an effort to figure out what is killing the antibodies, the amount of ratio of antibody to linker used was reduced and the incubation conditions were changed. The original ratio of antibody to linkers was approximately 1:10,000 which was an extreme excess. These conditions seem to be better for the antibodies, but no significant cage binding was seen in any of the samples. The next step is to confirm that there is DNA bound to the antibodies or if it a problem with the DNA cages themselves.