Protocol for synthesizing and purifying short-length poly(ADP)ribose polymer using fast protein liquid chromatography

Here, we present a protocol for synthesizing and purifying high-yield, short-length poly(ADP)ribose (PAR) polymers using fast protein liquid chromatography (FPLC). We describe steps for expressing and purifying proteins, in vitro synthesis of PAR, and fractionation and visualization of PAR. This cost-effective, non-hazardous, shorter approach avoids the use of radiolabeled substrates, hazardous reagents, and specialized equipment, producing homogenous PAR chains under 10 units. It eliminates the need for enzymes such as PARG and SVP, enabling broad accessibility for biophysical and structural studies. • Steps for purifying human PARP1 through immobilized metal affinity chromatography • Procedure for synthesis of poly(ADP)ribose • Guidance on fractionation of poly(ADP)ribose using Resource Q column • Instructions for purification of short-length PAR Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for synthesizing and purifying high-yield, short-length poly(ADP-ribose) (PAR) polymers using fast protein liquid chromatography (FPLC). We describe steps for expressing and purifying proteins, in vitro synthesis of PAR, and fractionation and visualization of PAR. This cost-effective, non-hazardous, shorter approach avoids the use of radiolabeled substrates, hazardous reagents, and specialized equipment, producing homogenous PAR chains under 10 units. It eliminates the need for enzymes such as PARG and SVP, enabling broad accessibility for biophysical and structural studies.