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

TL;DR

This paper provides a safe and cost-effective protocol for making short-length PAR polymers using FPLC, avoiding hazardous materials and specialized equipment.

Contribution

A new, accessible method for synthesizing and purifying short-length PAR without radiolabeled substrates or specialized enzymes.

Findings

The method produces homogenous PAR chains under 10 units using FPLC.

It avoids the need for PARG and SVP enzymes, simplifying the process.

The approach is cost-effective and suitable for biophysical and structural studies.

Abstract

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

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.

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