A detailed protein-SELEX protocol allowing visual assessments of individual steps for high success rate.

As a nucleic acid alternative to traditional antibody, aptamer holds great potential in various fields of biology and medicine such as targeted gene therapy, drug delivery, bio-sensing and laboratory medicine. Over the past decades, conventional SELEX method has undergone dramatic modifications and improvements owing to developments in material sciences and analytical techniques. However, many of the recently developed strategies either require complex materials and instruments or suffer from low efficiency and high failure rates in the selection of desired aptamers. Accordingly, the development of aptamers against new or novel targets is still a major obstacle for aptamer-based research and application. Here, we present an improved protein-SELEX procedure for simplified and highly efficient isolation of aptamers against protein targets. We describe approaches that ensure a high success rate of aptamer selection via simplifying PCR procedures, introducing denature gel, utilising an electro-elution based ssDNA separation strategy as well as an ELISA-based highly sensitive binding assay. In addition, a simplified sample preparation method for MiSeq-based next generation sequencing is also introduced. While a recombinant protein as a bait for SELEX is discussed here, this protocol will also be invaluable for researchers wishing to develop aptamers against targets other than proteins such as small molecules, lipids, carbohydrates, cells and micro-organisms for future gene therapy and/or diagnostics.