Rho1D4 Affinity Resins

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 affinity Agarose from Cube Biotech was successfully used in the following publications:

Protein	Year	Author
Rhodopsin Mutants	2015	Mattle D., Singhal A., Schmid G., Dawson R., Standfuss J., ¹

Features

Usage	Specific binding and purification of Rho1D4-tagged proteins
Specifity	Affinity to Rho1D4-tagged proteins
Binding capacity	>3 mg/mL
Stability	Highly tolerant to detergents
Filling quantity	Delivered as a 50 % suspension
Bead size	40 μm
Bead Ligand	Rho1D4 antibody
Required equipment	Lysis Buffer Wash Buffer Elution Buffer Rho1D4 peptide, this is part of the Rho1D4 starter set or can be aquired separately Detergents Ultrasonic homogenizer Ice bath Refrigerated centrifuge for 50 mL tubes (min10,000 x g) and 2 mL tubes Refrigerated superspeed or ultracentrifuge capable of 100.000 x g End-over-end rotator 2 mL microcentrifuge tubes 15 mL polypropylene tube, 50 mL polypropylene tube and 50 mL polycarbonate high speed centrifuge tube (for ultracentrifuge) Micropipettor and Micropipetting tips Disposable gravity flow columns with capped bottom outlet, 2 ml  pH meter End-over-end shaker SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment

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.

Applications

All protocols and buffer compositions are also avaible as PDF-Files on the Protocols & Datasheets page.

A.	Solubilization of the membrane protein:

	Thaw the E. coli cell pellet on ice for 15 min. Optional: Freezing the cell pellet at -20 °C for 30 min prior to incubation at room temperature improves lysis by lysozyme. Resuspend the cell pellet in Lysis Buffer. Use 10 mL Lysis Buffer per g cell pellet. Pour it into a 50 mL conical centrifuge tube. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption. Note: Keep the lysates on ice to prevent warming. Incubate on an end-over-end shaker at 4 °C for 1 h. Centrifuge the lysate for 15 min at 900 x g and 4 °C to remove cell debris. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis. Carefully transfer the supernatant to a fresh tube. Centrifuge for 30 min at 7,000 x g and 4 °C to precipitate inclusion bodies. Tip: Analyze the resulting pellet by SDS-PAGE to assess if target protein is present in inclusion bodies. To capture these proteins, we recommend purification via His-tag under denaturing conditions, using PureCube Ni-NTA Agarose. Alternatively, optimize expression conditions to bring the target protein into the membrane fraction Carefully transfer the supernatant to a polycarbonate high-speed centrifuge tube and centrifuge at 100,000xg for 1 h at 4 °C. Discard the supernatant and resuspend the pellet in 5 mL EW Buffer. Determine protein concentration and adjust the volume with EW Buffer to a concentration of 5 mg/mL. Note the adjusted volume. Note: The solution contains the total membrane protein fraction. Based on the results from the detergent screen, calculate the amount of detergent needed to solubilize the protein in the adjusted volume. Add the detergent. Note: To determine optimal detergent conditions, refer to the Cube Biotech Protocol: "Screening Detergents for Optimal Solubilization and Purification of Membrane Proteins" Transfer the suspension to a clean 15 mL polypropylene centrifuge tube. Incubate on an end-over-end rotator using the incubation conditions determined in the detergent screen. Transfer the suspension to a polycarbonate high-speed centrifuge tube and centrifuge at 100,000 x g for 1 h at 4 °C Transfer the supernatant to a fresh 15 mL tube and use it in part B of the protocol. Note: The solution contains the solubilized membrane protein fraction. Resuspend the PureCube Rho1D4 Agarose by inverting the bottle until the suspension is homogeneous. Transfer 0.2 mL of the 50 % suspension (corresponding to 100 μL bed volume) to a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant.
B.	Purification of the membrane protein:
	Add 1 mL EW Buffer and gently resuspend the slurry to equilibrate the resin. Allow the resin to settle by gravity and remove the supernatant. Tip: Alternatively, resin equilibration can be performed directly in the disposable gravity flow column. Pipet the soluble membrane fraction onto the equilibrated PureCube Rho1D4 Agarose and incubate at 4˚C overnight on an end-over-end shaker. Transfer the binding suspension to a disposable gravity flow column with a capped bottom outlet. Use EW Buffer to rinse the centrifuge tube and remove resin adhered to the wall. Remove the bottom cap of the column and collect the flow-through. Wash the column with 0.5 mL EW Buffer. Repeat the washing step at least 3 times. Elute the rho1D4-tagged protein by adding 0.2 mL Elution Buffer. Close and rotate the column for 1 h at 4°C. Remove the top and bottom cap of the column and collect the eluate. Repeat step 7 at least 5 times. Collect each eluate in a separate tube and determine the protein concentration of each fraction. Analyze all fractions by SDS-PAGE and Bradford assay or spectrophotometry (280 nm). Note: Do not boil membrane proteins. Instead, incubate samples at 46˚C for 30 min in preparation for SDS-PAGE analysis. Optional: Perform a Western Blot assay using Rho1D4 antibody.

References

1. Mattle D. et al.(2015). Mammalian Expression, Purification, and Crystallization of Rhodopsin Variants. In: Jastrzebska B. (eds) Rhodopsin. Methods in Molecular Biology, vol 1271. Humana Press, New York, NY