Rho1D4 protein purification in detail
Fig. 1: Purification of chemokine receptor 4 (CXCR4) using PureCube Rho1D4 Agarose. Total E.coli lysate (TL) was solubilized with Fos-Choline-14 and the soluble fraction (SF) was incubated on an immunoaffinity column loaded with rho1D4 antibody. Wash fractions (W1-W3) show no detectable loss of target protein. Concentration of eluted CXCR4 in elution fractions (E1-E4) ranged 0.6-1.0 mg/mL as determined spectrophotometrically.
Purifies protein with high specificity and yield
PureCube resins are produced under strict quality guidelines and each batch undergoes quality checks to ensure that the loaded matrix has a high protein capacity. Combined with the specificity of the antibody-epitope interaction, a purification protocol optimized for the target protein can generate elution fractions with exceptionally high yields. Figure 1 shows a purification run for chemokine receptor 4 (CXCR4). The tagged protein was expressed in E. coli, solubilized with Fos-Choline®-14 and purified on a column containing PureCube Rho1D4 Agarose beads. Using the rho1D4 peptide as eluent, the 4 elution fractions contained a total recoved protein concentration of 0.8, 1.0, 0.85 and 0.6 mg/mL. A western blot shows CXCR4 at approximately 65 kDa and 35 kDa. These bands represent dimers and monomers of the 39.7 kDa membrane protein. Separation of monomers and dimers, as well as removal of the eluent peptide, can be done with size-exclusion chromatography.
Pure and active membrane proteins
Purification results for membrane proteins purification like this can not be archived by using more common resins like Ni-NTA. Therefore Rho1D4 is your way of choice here.
Fig. 2: Coomassie blue stains of sucessfull membrane protein purifications afther using the Rho1D4 tag. A: Protein monomere of ~65kDa. B: Tetrameric protein of ~135 kDA. C: Heterodimieric protein, both subunity can be seen and a smaller nanodisc complex and has been used to stabilize the protein.
Rho1D4 Agarose was successfully used in the following publication:
Mattle, D. et al. (2015) Mammalian Expression, Purification, and Crystallization of Rhodopsin Variants. Beata Jastrzebska (ed), Rhodopsin: Methods and Protocols. Methods in Molecular Biology, vol. 1271, Chapter 3, DOI 10.1007/978-1-4939-2330-4_3, Springer Science +Business Media New York