Rho1D4 tag / 1D4 tag

Rho1D4, also called "1D4" is an affinity tag consisting of nine amino acids (T-E-T-S-Q-V-A-P-A). The tag is preferrably added to the C-Terminus of a protein of interest. Agarose Resins and magnetic beads coupled with the matching Rho1D4-antibody are able to purify proteins that are tagged with Rho1D4. Rho1D4 protein purification assays combine the incredible high specificity of other antibody based protein purification with an high protein yield. For more information regarding Rho1D4 we have written this guide to provide you with all necessary information.

Overview of Cube Biotech's Rho1D4 affinity products for protein purification.


Rho1D4 agarose resin (40 µm)	Rho1D4 agarose (40 µm) Cartridge	Rho1D4 MagBeads (30 µm)
Rho1D4 agarose resin (XL sized)	Rho1D4 agarose (40 µm) COMPACT Cartridge	Rho1D4 MagBeads (XL sized)
	Rho1D4 starter sets - For all who want to try out the Rho1D4 tag for the first time
Starter Set 1 (1ml agarose + 5mg peptide)	Starter Set 3 (1 x 1ml Cartridge + 5mg peptide)	Starter Set 2 (1ml MagBeads + 5mg peptide)
	Starter Set 4 (1 x 1ml Compact Cartridge + 5mg peptide)
	Rho1D4 protein purification support products
Rho1D4 peptide (for protein elution)	Rho1D4 tagged GFP (for positive controls)	Rho1D4 antibody [mouse] (for blots)

Features

Usage	Specific binding and purification of Rho1D4 - tagged proteins
Specifity	Affinity to Rho1D4-tagged proteins
Stability	Highly tolerant to detergents
Bead Ligand	Rho1D4 antibody
Protein Yield	3-4 mg/ ml resin or MagBeads

Rho1D4 products by Cube Biotech were used in the following publications:

Protein	Year	Author	Rho1D4 product
Rhodopsin Mutants	2015	Mattle D., Singhal A., Schmid G., Dawson R., Standfuss J., ¹	Rho1D4 agarose (40µm)
PRG2	2019	Brosig A., Fuchs J., Ipek F., Kroon C., Schrötter S., Vadhvani M., Polyzou A., Ledderose J., van Diepen M., Holzhütter H-G., Trimbuch T., Gimber N., Schmoranzer J., Lieberam I., Rosenmund C., Spahn C., Scheerer P., Szczepek M., Leondaritis G., Eickholt B.J.²	Rho1D4 agarose (40µm)
Opsin	2019	Ahmed C. M., Dwyer B. T., Romashko A., Van Adestine S., Park E.-H., Lou Z., Josiah S., Savinainen A., Zhang B., Lewin A.S.³	Rho1D4 agarose (40µm)
Visual Pigments / Opsins	2018	Liu D-W., Wang F_Y., Lin J-J., Thompson A., Ying L., Vo D., Yan H.Y., Zakon H.H., ⁴	Rho1D4 MagBeads (30µm)
P2X4	2019	Kotov V., Bartels K., Veith k., Josts I., Subhranabyam U. K. T.,. Güther C., Labahn., Marlovits T.C., Moraes I., Tidow H., Löw C., Garcia-Alai M.M.⁵	Rho1D4 MagBeads (30µm)

Rho1D4 protein purification in detail

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.

Fig. 2: 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.

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. 1: 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 n

References:

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
Brosig, A. et al (2019). The Axonal Membrane Protein PRG2 Inhibits PTEN and Directs Growth to Branches. Cell Reports. 29. 2028-2040.e8. 10.1016/j.celrep.2019.10.039.
Ahmed, Chulbul M., et al. "SRD005825 Acts as a Pharmacologic Chaperone of Opsin and Promotes Survival of Photoreceptors in an Animal Model of Autosomal Dominant Retinitis Pigmentosa." Translational Vision Science & Technology 8.6 (2019): 30-30.
Liu, Da-Wei et al. (2018). The Cone Opsin Repertoire of Osteoglossomorph Fishes: Gene Loss in Mormyrid Electric Fish and a Long Wavelength-Sensitive Cone Opsin That Survived 3R. Molecular Biology and Evolution. 10.1093/molbev/msy241.
Kotov, Vadim et al. (2019). High-throughput stability screening for detergent-solubilized membrane proteins. Scientific Reports. 9. 10.1038/s41598-019-46686-8.

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