Detection of ADP ribosylation in PARP-1 and bacterial toxins using a capillary-based western system

Abstract

Both poly and mono ADP-ribosylation are common posttranslational protein modifications. For example, poly ADP-ribosylation is involved in DNA repair mechanisms through the poly (ADP-ribose) polymerase (PARP) family of enzymes. While mono ADP-ribosylation has been known to trigger cell death exhibited by many bacterial toxins. Because of the wide role of ADP-ribosylation, the detection and analysis are very important for further understanding of the PARP family of enzymes and the molecular mechanisms leading to cell toxicity in the presence of bacterial enzymes. Here, we describe a novel technique utilizing a CE-based Western technology to detect and analyze ADP-ribosylated proteins. The method is based on a nanovolume size separation that is automated, quantitative, offers great sensitivity, and is high-throughput for potential use in PARP drug screening inhibitor assays.

Keywords: ADP-ribosylation; Capillary Western; Clostridium difficile binary toxin; Diphtheria toxin; PARP-1.