Purification of rho1D4-tagged Membrane Proteins Using PureCube Rho1D4 Agarose

 

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. With a binding capacity of 3-4 mg protein per mL resin, PureCube Rho1D4 Agarose is a high-quality affinity matrix designed for the efficient purification of rho1D4-tagged membrane proteins. This protocol is optimized for tagged proteins expressed in E. coli and a bed volume of 0.1-1 mL. It is possible to scale up the protocol for higher volumes. 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, 200 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
NaH2PO4 * 10 mM 119.98 0.5 M 29.99 g/ 500 mL 4 mL
NaCl* 150 mM 58.44 5 M 146.1 g/ 500 mL 6 mL
Glycerol 10 % (v/v) - 100 % - 20 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, 20 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
Rho Buffer 1x - 1x - 20 mL
Lysozyme 1 mg/mL - - - 20 mg
Instructions: Always prepare fresh.

Equilibration and Wash (EW) Buffer, 50 mL Note: Only Required when purifying membrane proteins

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
Rho Buffer 1x - 1x - 50 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

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock 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

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock 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.Solubilization of the membrane protein:
 
 
  1. 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.
  2. 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.
  3. 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.
  4. Incubate on an end-over-end shaker at 4 °C for 1 h.
  5. 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.
  6. 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
  7. Carefully transfer the supernatant to a polycarbonate high-speed centrifuge tube and centrifuge at 100,000xg for 1 h at 4 °C.
  8. 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.
  9. 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"
  10. 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.
  11. Transfer the suspension to a polycarbonate high-speed centrifuge tube and centrifuge at 100,000 x g for 1 h at 4 °C
  12. 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:
 
 
  1. 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.
  2. 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.
  3. Pipet the soluble membrane fraction onto the equilibrated PureCube Rho1D4 Agarose and incubate at 4˚C overnight on an end-over-end shaker.
  4. 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.
  5. Remove the bottom cap of the column and collect the flow-through.
  6. Wash the column with 0.5 mL EW Buffer. Repeat the washing step at least 3 times.
  7. 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.
  8. Repeat step 7 at least 5 times. Collect each eluate in a separate tube and determine the protein concentration of each fraction.
  9. 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.
  10. Optional: Perform a Western Blot assay using Rho1D4 antibody.
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