Getting Started with Rho1D4
Getting started with the Rho1D4 System
Using the rho1D4 system to purify membrane proteins requires some protocol optimization, as each protein behaves differently once expressed in a heterologous host, in response to solubilization detergents, and within the purification column. Important is to have all the items necessary and to screen for optimal protocol conditions and steps. Below are the 4 items needed and each is accompanied by protocols or information to help you implement the rho1D4 system quickly and efficiently. If we can help with the optimization, please contact us.
Your target protein needs to be fused to the rho1D4-tag. You can do this on the C- or on the N-terminus, depending on where the tag interferes the least with protein activity. These protocols outline primer design (with copy-paste sequences) for the necessary expression constructs.
Prokaryotic and eukaryotic systems offer different advantages when expressing your target protein. Selection of an appropriate expression system requires some trial-and-error and screening for optimal expression conditions. This protocol details the optimization of host, media, temperature, and induction time for protein expression in bacterial systems (E. coli).
Detergents are used to solubilize membrane proteins and selecting the optimal detergent for your target protein is critical for subsequent purification. This protocol outlines the screening of 7 commonly used detergents.
An affinity matrix coupled to the rho1D4 antibody and the rho1D4 peptide to serve as a competitive eluent are needed to purify your rho1D4-tagged protein. PureCube Rho1D4 Agarose and Rho1D4 peptide can be purchased together as a starter set or individually to meet your throughput needs. For detection in Western Blots, a highly specific Rho1D4 antibody is available. This protocol outlines the solubilization and purification process.