Protocol for preparing Thiopropyl Sepharose resin used for capturing S-nitrosylated proteins

Abstract

Summary: S-nitrosothiol (SNO)-Resin Assisted Capture (SNO-RAC) relies on a Thiopropyl Sepharose resin to identify S-nitrosylated proteins (SNO-proteins) and sites of S-nitrosylation. Here, we present a protocol for preparing Thiopropyl Sepharose resin with efficiency of SNO-protein capture comparable to the discontinued commercial version. We describe steps for amine coupling, disulfide reduction, and generation of thiol reactive resin. We then detail quality control procedures. This resin is also suitable for Acyl-RAC assays to capture palmitoylated proteins.For complete details on the use and execution of the SNO-RAC protocol, please refer to Forrester et al.,1 Fonseca et al.,2 and Seth et al.3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Signal Transduction
• Protein Biochemistry
• Chemistry