Assessment of commercial versus custom-made MuRF1 antibodies

Dawson, Peter William James (2020). Assessment of commercial versus custom-made MuRF1 antibodies. University of Birmingham. M.Sc.

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Abstract

Muscle wasting is the result of muscle protein breakdown occurring at a greater rate than muscle protein synthesis, resulting in a net loss of muscle mass. Muscle protein breakdown is contributed to by the ubiquitin proteasome system, where E3 ubiquitin ligases facilitate the degradation of proteins; MuRF1 has been identified as an E3 ubiquitin ligase that targets muscle proteins as a substrate. The role of MuRF1 function is still under investigation and there is no published data on the efficacy of immunoblotting based techniques used to identify this protein. The aim of this study was to compare a novel custom-made vs commercial MuRF1 antibodies in for the purpose of western blots and immunoprecipitation. Preliminary dot blots showed that rabbit sera stimulated to produce MuRF1 antibodies were able to detect MuRF1 specific peptides. Western blots revealed that purified custom-made antibodies were able to detect recombinant MBP-MuRF1 but not MuRF1 in overexpressed cell lysate; Santa Cruz offered the most sensitive and specific commercial MuRF1 antibody for western blot. Custom-made antibodies were the most effective at enriching MuRF1 in immunoprecipitation. This provides an important set of tools to characterise MuRF1, explore muscle atrophy signalling, and produce therapies to treat chronic muscle pathologies.

Type of Work:

Thesis (Masters by Research > M.Sc.)

Award Type:

Masters by Research > M.Sc.

Supervisor(s):