Purification of rho1D4-tagged Membrane Proteins Using PureCube Rho1D4 Magbeads

Tagging a membrane protein with the rho1D4 epitope to purify on an immunoaffinity matrix loaded with the rho1D4 antiobdy has proven to be an effective purification method for membrane proteins. Once the process is optimized, pure protein fractions (>85% purity) can generally be obtained. PureCube Rho1D4 MagBeads are well-suited to purify rho1D4-tagged membrane proteins from dilute solutions, such as cell culture or medium supernatants. This protocol is optimized for tagged proteins expressed in E. coli culture volumes of 20-60 mL and a MagBead volume of 200 µL. It is possible to scale up and down the protocol. The rho1D4-tagged target protein is purified from the cleared lysate under native conditions in a bind-wash-elute procedure. Binding is performed in batch mode (as opposed to on-column binding). This method is most efficient, especially when the target protein is present at low concentrations. This procedure should be preceeded with screens for an optimal expression system and solubilization detergent. Cube Biotech provides general detergent screen protocols (www.cube-biotech.com/protocols). Also note that if the expressed protein is found mainly in inclusion bodies, it may be preferable to purify the protein on PureCube His Affinity matrices under denaturing conditions.

Buffer compositions

Rho Buffer, 100 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for stock	Stock needed for buffer

NaH₂PO₄ *	10 mM	119.98	0.5 M	29.99 g/ 500 mL	2 mL
NaCl*	150 mM	58.44	5 M	146.1 g/ 500 mL	3 mL
Glycerol	10 % (v/v)	-	100 %	-	10 mL
Protease inhibitor	1x	-	100 %	-	follow supplier‘s instructions
Instructions: Mix in 160 mL water. Adjust the pH to 7.0 using NaOH and then add water to a total volume of 200 mL. Add protease inhibitor directly before use. *Note: Depending on the protein purified, PBS at pH 7.4 may yield better results.

Lysis Buffer, 10 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for stock	Stock needed for buffer

Rho Buffer	1x	-	1x	-	10 mL
Lysozyme	1 mg/mL	-	-	-	10 mg
Instructions: Always prepare fresh.

Equilibration and Wash (EW) Buffer, 30 mL Note: Only Required when purifying membrane proteins

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for stock	Stock needed for buffer

Rho Buffer	1x	-	1x	-	30 mL
Detergent	based on screen**	-	-	-	based on screen**
Instructions:Always prepare fresh **Typically 1.5-2x critical micellar concentration (CMC) of detergent. Use the detergent that yielded the best solubilization results in the detergent screen; see Cube protocol „Screening Detergents for Optimal Solubiization and Purification of Membrane Proteins“ .

Elution Buffer, 10 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for stock	Stock needed for buffer

Rho Buffer	1x	-	1x	-	50 mL
Detergent	based on screen**	-	-	-	based on screen**
Rho1D4 peptide^§	200 µM	947	10 mM	5 mg / 530 µL ddH₂O	200 µL
Instructions:Always prepare fresh **Typically 1.5-2x critical micellar concentration (CMC) of detergent. Use the detergent that yielded the best solubilization results in the detergent screen; see Cube protocol „Screening Detergents for Optimal Solubiization and Purification of Membrane Proteins“ . § The recommended concentration of Rho1D4 peptide in the elution buffer is 200 µM-1 mM. See the Rho1D4 peptide Datasheet for further instructions to recontitute the lyophilized peptide.

5X SDS Page Buffer, 10 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for stock	Stock needed for buffer

Tris-HCl, pH 6.8–7.0	300 mM	121,14	1 M	121,14 g/ 1 L	3 mL
Glycerol	50% (v/v)	-	100% (v/v)	-	5 mL
SDS	5% (w/v))	-	-	-	0.5 g
Bromophenol blue	0.05% (w/v)	-	4%	-	125 μL
DTT	250 mM	154.25	1 M	1.54 g/ 10 mL	125 μL/aliquot
Instructions: Make sure to prepare a 1 M Tris-HCl stock by dissolving Tris base in 500 mL deionized water, adding HCl to a pH of 6.8–7.0, and adding water to a final volume of 1 L. For the SDS-PAGE Buffer, mix all components listed except DTT and add water to a total of 10 mL. Freeze 20 aliquots (375 µL each) at –20˚C. Before use, add DTT to the needed single aliquots.

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.