Rho1D4 Affinity Resins

PureCube Rho1D4 Agarose

Proteins tagged with the epitope sequence of the rho1D4 antibody can be purified with this affinity matrix. Based on BioWorks Workbeads, Cube Biotech produces the first commercially available immunoaffinity resin for the Rho1D4 purification system. Rho1D4 peptide in the Elution Buffer serves to competitively bind to the affinity resin and release the tagged protein, providing gentler elution conditions than, for example, changing pH. Cube Biotech offers conveniently aliquoted and priced Rho1D4 peptide that has been tested with PureCube Rho1D4 Agarose. The peptide can be purchased individually, or together with PureCube Rho1D4 Agarose as part of a Starter Set. For detection in Western Blots, a highly specific Rho1D4 antibody is available. For dilute samples or pull-down experiments, we recommend using PureCube Rho1D4 MagBeads.

The Rho1D4 system provides:

ideal solution for membrane protein research
binding capacity of up to 3-4 mg protein per mL resin
highly specific purification
gentle protein elution based on competitive binding
Also available as MagBeads and prepacked cartridges

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

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