The rho1D4 purification system is based on the highly specific binding of the rho1D4 antibody to the rho1D4 epitope fused to proteins. PureCube Rho1D4 MagBeads are ferrimagnetic agarose beads loaded with the rho1D4 antibody; a system that is particularly useful for dilute samples and pull-down experiments. The eluent peptide can be purchased individually
, or together with PureCube Rho1D4 MagBeads as part of a Starter Set. PureCube Rho1D4 MagBeads are delivered as a 5% suspension. For detection in Western Blots, a highly specific Rho1D4 antibody
PureCube Rho1D4 MagBeads provide:
- A dedicated purification solution for membrane proteins
- Binding capacity of up to 3 mg protein per mL settled beads
- Purification of proteins expressed at low levels or from diluted solutions
- Low unspecific binding
- Rho1D4 Agarose also available
Rho1D4 MagBeads from Cube Biotech were successfully used in the following publications:
|Visual Pigments / Opsins
||Liu D-W., Wang F_Y., Lin J-J., Thompson A., Ying L., Vo D., Yan H.Y., Zakon H.H., 1
||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.2
What is Rho1D4? - An antibody specialized in membrane protein purification
Rho1D4 is the name for the last nine amino acids of the intracellular C-terminus of the bovine rhodopsin protein. The name originates from a monoclonal antibody that specifically binds to this nine amino acids long sequence. Together with said antibody, this tag can be used for highly specific protein purification, especially for membrane proteins like GPCRs. A (membrane) protein of interest can be modified to incorporate this epitope at its C-Terminus (Fig 1.) Subsequently the protein of interest can be purified using an affinity matrix that has the Rho1D4 antibody coupled to it. The following elution is archived by adding an excessive amount of the Rho1D4 peptide
to compete with the tagged protein of interest over the binding to the antibody. This ensures a gentler elution procedure than for example pH changes that are applied in other purification methods. Learn more.
Fig. 1: A schematic depiction of a membrane protein with an C-terminally added Rho1D4 tag. The tag's sequence T-E-T-S-Q-V-A-P-A contains only nine amino acids and is located in the ECM.
||Specific binding and purification of Rho1D4 - tagged proteins
||Affinity to Rho1D4-tagged proteins
||3mg/mL settled beads
||Highly tolerant to detergents
||Delivered as a 5 % suspension
- Lysis Buffer
- Wash Buffer
- Elution Buffer
- Rho1D4 peptide, this is part of the Rho1D4 starter set or can be aquired separately
- Ultraconic homogenizer
- Ice bath
- Refrigerated centrifuge (min 10,000 x g) for 1,5 ml tubes
- Ultraspeed Refrigerated centrifuge capable of 100,000 x
- End-over-end rotator
- 2 mL microcentrifuge tubes
- Polycarbonate high speed centrifuge tube
- Micropipettor and Micropipetting tips
- Magnetic holder for microcentrifuge tubes (for separation of magnetic beads)
- pH meter
- UV/VIS spectrophotometer
- SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment
|A.||Protocol for Solubilization of membrane proteins|
- 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 200 μL Lysis Buffer per 20 mg cell pellet. Pour it into a microcentrifuge 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 consitions, using PureCube His Affinity matrices. 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 200 μL 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 our protocols page.
- Transfer the suspension to a clean microcentrifuge 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 microcentrifuge tube and use it in part B of the protocol. Note: The solution contains the solubilized membrane protein fraction.
| B.||Purification of the membrane protein using Rho1D4 MagBeads|
- Resuspend the PureCube Rho1D4 MagBeads by vortexing. Transfer 0.2 mL of the 5% suspension into a 1.5 mL microcentrifuge tube. Note: PureCube Rho1D4 MagBeads are supplied as a 5% suspension with a binding capacity of about 15-20 µg/mL. If you expect a higher protein concentration in your sample, increase the suspension volume.
- Add 0.5 mL EW Buffer and mix gently. Place the tube on a magnetic microtube stand until the beads are separated and remove the supernatant. Repeat once.
- Pipet the soluble membrane fraction onto the equilibrated PureCube Rho1D4 MagBeads and incubate at 4˚C overnight on an end-over-end shaker.
- Place the tube on a magnetic microtube stand until the beads are separated and remove the supernatant.
- Remove the tube from the magnet. Add 0.5 mL of Buffer EW and mix gently. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
- Repeat the wash step at least twice.
- Elute the Rho1D4-tagged protein by adding 100 µL Elution Buffer. Close and rotate the column for 1 h at 4°C. Place the tube again on the magnetic microtube stand and allow the beads to separate. Collect the supernatant.
- 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.
1. 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.
2. Kotov, Vadim et al. (2019). High-throughput stability screening for detergent-solubilized membrane proteins. Scientific Reports. 9. 10.1038/s41598-019-46686-8.