Rho1D4 tag / 1D4 tag

Content:

2. Rho1D4-tag protein purification products

3. Publications using the Rho1D4-tag

4. Rho1D4-tag protein purification in use

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.

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
Tag Sequence	TETSQVAPA

Why Cube Biotech's Rho1D4-tag protein purification products?
There are numerous reasons to choose Cube Biotech. Here are a few.
Size of the tag 902.9 Da 9 amino acids (TETSQVAPA)	Protein yield per ml resin 3-4 mg/ml	Specificity of interaction KD: 20 nM (5)	Elution conditions Rho1D4 peptide, low pH, or tag cleavage

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)
UPDATE OCTOBER 2022: Rho1D4 starter sets - For all who want to try out the Rho1D4-tag for the first time. Now on 50% discount!
Starter Set 1 (1 mL agarose + 5 mg peptide)	Starter Set 3 (1 x 1 mL Cartridge + 5 mg peptide)	Starter Set 2 (1 mL MagBeads + 5 mg peptide)
	Starter Set 4 (1 x 1 mL 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)

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)
NeoR	2020	Broser M., Spreen A., Konold P.E., Peter E., Adam S., Borin V., Schapiro I., Seifert R., Kennis J.T.M., Sierra Y.A.B., Hegemann P.⁶	Rho1D4 Agarose (40µm)
Gαs	2021	Swonger Koretz K., McGraw C.E., Stradley S., Elbaradei A., Malstadt N., Robinson A.S.⁷	Rho1D4 Agarose (40µm)
emST	2021	Bradberry M.M., Mishra S., Zhang Z., Wu L., McKetney J.M., Vestling M.M., Coon J.J., Chapman E.R⁹	Rho1D4 agarose Rho1D4 antibody Rho1D4 peptide
Immunoisolation of synpatic vesicles	2022	Weihou G.⁸	Rho1D4 agarose Rho1D4 antibody Rho1D4 peptide
	2022	Bradberry M.M., Mishra S., Zhang Z., Wu L., McKetney J.M., Vestling M.M. Coon J.J., Chapman E.R.⁹	Rho1D4 agarose Rho1D4 peptide
	2022	Rozenberg A., Kaczmarczyk I., Matziv D., Verock J., Nagata T., Sugiura M., Katayama K., Kawasaki Y., Konno M., Nagasaka Y., Aoyama M., Das I., Pahima E., Church J., Adam S., Borin V.A., Chazan A., Augustin S., Wietek J., Diene J., Peleg Y., Kawanabe A., Fujiwara Y., Yizhar O., Sheves M., Schapiro I., Furutani Y., Hideki K., Keeichi I., Hegemann P., Béjà O., Shalev-Benami¹⁰	Rho1D4 agarose

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 purified elution fractions of three different membrane proteins, two membrane proteins stabilized in detergents and the third in MSP1D1 nanodisc. The Rho1D4 tagged proteins were expressed in T.ni insect cells or Hek293 cells, respectively and purified on a column containing PureCube Rho1D4 Agarose beads. The Rho1D4 peptide was used as mild eluent. These bands represent membrane proteins corresponding to monomers, tetramers and heterodimers stabilized in nanodisc. Analysis of the oligomeric state 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 achieved by using more common resins like Ni-NTA. Therefore Rho1D4 is your way of choice here.

Fig. 1: Coomassie blue stains of successful membrane protein purifications after using the Rho1D4-tag.
A: Protein monomer of ~65kDa.
B: Tetrameric protein of ~135 kDA.
C: Heterodimieric protein, both subunits can be seen. The smallest band corresponding to ~ 28 kDa is a MSP1D1 nanodisc protein used for membrane protein stabilization.

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.
Broser, M. et al., (2020) NeoR a near-infrared absorbing rhodopsin. Nat Commun 11, 5682. https://doi.org/10.1038/s41467-020-19375-8
Swonger Koretz. et al., (2021) Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance. Biophysical Journal,. https://doi.org/10.1016/j.bpj.2021.02.032.
Weihou Guo (2021). Overexpression and isolation of the intermediate state of serotonin transporter from Echinococcus multilocularis –‒ the ER localized HSP complexes of the folding trajectory. PhD, University of Hamburg.
Bradberry et al. (2022). Rapid and gentle immunopurification of brain synaptic vesicles. The Journal of Neurosciences https://doi.org/10.1523/JNEUROSCI.2521-21.2022
Rozenberg et al. (2022). Rhodopsin-bestrophin fusion proteins from unicellular algae form gigantic pentameric ion channels. Nature structural & molecular biology https://www.nature.com/articles/s41594-022-00783-x