UC San Diego

Antibodies are widely used in various research applications, generally used to detect a protein of interest. The SETD5 gene is implicated in Autism Spectrum Disorder (ASD) but published literature is in disagreement on the molecular role of the SETD5 protein. Aiming to understand the molecular role in our systems, we used a polyclonal SETD5 antibody from Invitrogen by ThermoFisher Scientific (PA-53718) listed as approved for use in western blotting. Our SETD5 western blotting results showed multiple bands, including one that was in agreement with the predicted size of SETD5, 150 kDa, and another band at 60 kDa, that was concordant with the protein defined by ThermoFisher as a correct SETD5 band. In my correspondence with ThermoFisher, they were not able to provide adequate clarification on this inconsistency, and our effort prompted their leadership to remove the antibody from their website and catalog. This experience provided an example of the limitations with current antibody-based approaches such as western blotting or other protein quantification tools. To overcome this challenge, we are developing a novel protein quantification method called western-seq where the qualitative size information of western blotting is merged with quantitative DNA sequencing data, using antibodies that are conjugated to oligonucleotides. I focused on identifying optimal conditions for using conjugated antibodies. I identified a specific lysis buffer, sonication conditions, and single stranded binding protein (SSB) improved the signal to noise ratio when using conjugated antibodies for western blots. The western-seq system was first validated using a small set of conjugated antibodies with recombinant protein complexes, and then tested in HEK293 cells. We noticed that direct PCR amplification of membrane pieces provided the optimal conditions for generating sequencing libraries. Western-seq works as a functional protein quantification technique, and further optimization will enable western-seq to be a highly scalable and essential protein quantification method