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 using Rho1D4 Agarose
- 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.