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 |
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B. | Purification of the membrane protein using Rho1D4 MagBeads |
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