Blog post by Dr. Tamara Maiuri
As mentioned in the year-end review post, there were a few experiments I forgot to deposit to Zenodo.
Here is one: optimizing the method of detecting poly-ADP ribose (PAR) by immunofluorescence. Nothing ground-breaking in this report, but it was the key to detecting the increased PAR levels in HD patient fibroblasts.
Trying to detect PAR by immunofluorescence was very frustrating until I went to the CSHL meeting on The PARP Family and ADP-ribosylation last year. There I met Dr. Hana Hanzlikova, previously from Prof. Keith Caldecott’s lab at University of Sussex and now at the Institute of Molecular Genetics of the ASCR in Prague.
Dr. Hanzlikova gave me two important tips: you need to block the degradation of PAR using PARG inhibitors (because PAR turnover is too fast to catch it in action), and the best detection reagent is MABE1016, a macro domain fused to rabbit Fc tag. Sure enough, these conditions allowed us to visualize PAR formation in response to oxidative stress. The conditions and results have now been deposited to Zenodo.