Careful choice of detergents is maybe the most crucial factor when handling membrane proteins. Membrane proteins are in contact with lipidic membranes via large hydrophobic regions. Their extraction requires the use of detergents which possibly interfere with membrane protein structure and function. Different detergents can be optimal for extraction, purification, and crystallization steps. Cube Biotech offers a range of high quality, >99,5% pure, crystallization-grade detergents at an attractive price. Our detergents are delivered as convenient aliquots that keep fresh and provide optimal performance. Screening sets provide easy testing of a range of detergents. Please
contact us if you are interested in bulk amounts or detergents not listed here.
Screening detergents for optimal solubilization and purification of membrane proteins
The hydrophobic domain of membrane proteins makes them difficult to solubilize from their anchor in cell membranes. Detergents are amphipathic molecules with a polar head and a long hydrophobic carbon chain that form micelles in which membrane proteins embed and thus, can remain in aqueous solution. The resulting proteindetergent complex can then be isolated and purified.
The selection of the right detergent is crucial for the effective purification of a membrane protein. Factors to bear in mind are: (1) the critical micelle concentration (CMC) of a detergent, or the concentration at which the detergent forms micelles and accumulates in the membrane; (2) that changing protein or salt concentration can change the aggregation behavior of a protein and the CMC of a detergent; (3) that prolonged exposure to a detergent can delipidate a protein which leads to instability; and (4) that certain detergents are suboptimal for subsequent chromatographic
This protocol delineates the screening of detergents to solubilize a membrane protein expressed in a bacterial host (see Protocol "Screening for Optimal Membrane Protein Expression Conditions Using E. coli Expression Systems"). The detergent concentrations provided for the solubilization and for buffers used in the purification protocol (Table 1) are a starting point and may need to be adjusted for the target protein. Likewise, parameters such as temperature and length of solubilization can also be optimized in this screen (Fig. 1). Results from the screen are analyzed by Western blot, using a detection antibody specific to the target protein or the fused tag (e.g., His-tag, GST-tag, Strep®-tag, Rho1D4-tag). The best detergent and solubilizing conditions, as determined with this screen, are then implemented for the purification protocol corresponding to the used affinity tag (www.cubebiotech. com/protocols).
| Equipment | Materials |
|---|
| - UV/VIS Spectrophotometer
- SDS-PAGE and Western blotting equipment
- pH-meter
- Ultrasonic homogenizer
- Waterbath
- Refrigerated tabletop centrifuge
- Endoverend rotator
- 2mL microcentrifuge tubes
| - Tris base
- Sodium chloride (NaCl)
- Glycerol
- Imidazole
- Hydrochloric acid (HCl)
- Sodium dodecyl sulfate (SDS)
- Bromophenol blue
- Dithiothreitol (DTT)
- 1 g pellet of expression host (eg., E. coli) that has
completed a round of protein expression under
optimized conditions (see Protocol "Screening
for Optimal Membrane Protein
Expression Conditions using
E. coli Expression Systems"). - Lysozyme
- Benzonase® Nuclease (Novagen #721063)
- Octylβglucopyranoside (OG, 1 g; Cube BIotech #16001)
- nOctyl1thioβDglucopyranoside (OTG, 1 g; Cube Biotech #16017)
- nDecylβmaltopyranoside (DM, 1 g; Cube Biotech #16009)
- nDodecylβmaltoside (DDM, 1 g; Cube Biotech #16013)
- FOScholine ®12 (FC12)
- N,NDimethyldodecylamineNoxide (LDAO, 1 g; Cube Biotech #16005)
- A detection antibody that binds to the target protein
or protein tag (for Western blot analysis). - Lysis buffer corresponding to used affinty tag.
|
Solutions and buffers
Lysis Buffer, 1000 mL
The lysis buffer to use depends on the target protein and the affinity tag. Cube Biotech provides in each tagspecific purification protocol the recipe for a Lysis Buffer that has been tested with the corresponding tag (e.g., His-tag, GST-tag, Strep®-tag, Rho1D4-tag; cubebiotech. com/protocols). This Lysis Buffer should be used as an initial buffer that can be adapted as needed for the target protein.
5X SDS-PAGE Buffer, 10 mL
| Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for final buffer |
|---|
| TrisHCl, pH 6.8–7.0 | 300 mM | 121.14 | 1 M | 121.14 g/1 L | 3 mL |
| Glycerol | 50 % (v/v) | - | 100 % | - | 5 mL |
| SDS | 5 % (v/v) | - | - | - | 0.5 g |
| Bromophenol blue | 0.05% (v/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 1M TrisHCl 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 DDT and add water to a total of 10 mL. Freeze 20 aliquots (0.5 mL each) at –20˚C. Before use, add DTT to the needed single aliquots.
Procedure
- Using the expression culture scaled up as the last step in Protocol "Screening for Optimal Membrane Protein Expression Conditions using E coli Expression Systems", harvest a 1 g cell pellet by centrifuging at 4,000 rpm and 4˚C for 20 min.
- Complete the Lysis Buffer corresponding to the affinity tag used by adding 1 mg lysozyme per mL buffer needed.
- Carefully pour off the supernatant from the cell pellet and resuspend in lysozymeLysis Buffer. Use 10 mL buffer per g cell pellet.
- Add 12 μL Benzonase® Nuclease per 10 mL Lysis Buffer, and incubate the resuspended cells for 30 min at room temperature (15–25˚C).
- Disrupt cells by sonicating in short bursts for 3 min. Repeat once
- Centrifuge the lysate at 900xg and 4˚C for 15 min to remove cell debris.
- Transfer the supernatant to a fresh tube and centrifuge at 7,000xg and 4˚C for 30 min to precipitate inclusion bodies.
- Transfer the supernatant to a fresh tube, mix briefly, and distribute evenly among the number of 2 mL microcentrifuge tubes needed to test the desired detergents and solubilization conditions.
- Centrifuge the screening samples at 20,000xg and 4˚C for 1 h.
- Discard the supernatants. Label each tube with the detergent and solubilization conditions to be test (e.g., see Fig. 1).
- Resuspend each pellet in 500 μL Lysis Buffer.
- Add detergent to each tube in the amount to be tested. The solubilization concentrations given in Table 1 serve as a starting point, but different concentrations should be screened to optimize the solubilization of the target protein.
- . Incubate the samples on an endoverend rotator. As a starting point, incubate for 1 h at room temperature (15–25˚C). However, other incubation times and temperatures should be screened to optimize the solubilization conditions (e.g., Fig. 1).
- Remove a 20 μL aliquot from each tube for later analysis. Store the aliquots in 5 μL 5X SDS-PAGE Buffer at –20˚C.
- Centrifuge the screening samples at 20,000xg and 4˚C for 1 h.
- Remove a 20 μL aliquot from each tube for later analysis. Store the aliquots in 5 μL 5X SDS-PAGE Buffer at –20˚C.
- Thaw all aliquots taken for analysis and heat for 30 min at 46˚C.
- Analyze the aliquots by SDS-PAGE and Western blot to identify the detergent and conditions that afforded the best solubilization. Use this detergent and incubation parameters for the purification of the target protein.
Table 1: Overview of available Cube Ultrapure Detergents with corresponding CMC, solubilization concentration, and concentration for the purification. The solubilization and purification concentrations are given as a starting point and should be optimized for each target protein. Other detergents can also be screened in a similar fashion. CMC: critical micellar concentration.
| Detergent | Molecular weight (g/mol) | CMC in H2O (mM) | Solubilization concentration | Amount in 500 μL Lysis Buffer | Concentration for purification* |
|---|
| OG | 292.37 | ~18–20(2) | 51 mM | 7.46 mg | 27–40 mM |
| LDAO | 229.40 | ~1–2(3) | 30 mM | 3.44 mg | 1.5–4 mM |
| DM | 482.56 | ~1.6(4) | 21 mM | 5.07 mg | 2.4–3.2 mM |
| DDM | 510.62 | ~0.17(5) | 20 mM | 5.11 mg | 0.26–0.34 mM |
| OTG | 308.44 | ~9(6) | 51 mM | 7.87 mg | 13.5–18 mM |
| FC12 | 351.50 | ~1.5(7) | 32 mM | 5.62 mg | 2.3–3 mM |
* Concentration of detergent to be used in the Elution Buffer and the Equilibration and Wash Buffer of the purification protocol.
Fig. 1: Generic screen setup for solubilization detergent, concentration, temperature, and time. 3 detergents, 2 concentrations, 3 temperatures, and 3 incubation times are screened with 54 screening samples to determine optimal solubilization conditions. RT: room temperature.
Fig. 2: Western blot analysis of an example screening of 7 detergents. Aliquots of membrane factions solubilized with 7 different detergents were analyzed via SDS-PAGE and Western blot using an antibody specific to the tag fused to the target protein. Detergents OG and NG are poor solubilizers for this protein.
References:
- . Spriestersbach, A., Kubicek, J., Schaefer, F., Block, H., and Maertens, B. 2011. Purification of His-tagged proteins. Methods Navigator
- Lorber, B., Bishop, J. B., and DeLucas, L. J. 1990. Purification of OctylβDguchopyranoside and Reestimation of its Micellar Size. Biochim Biophys Acta—Biomembranes 1023: 254–265.
- Herrmann, K. W. 1962. Nonioniccationic Micellar Properties of Dimethyldodecylamine Oxide. J Phys Chem 66: 292
- Alpes, H., Apell, H.J., Knoll, G., Plattner, H., and Riek, R. 1988. Reconstitution of Na+/K+ ATPase into Phosphatidyl Choline Vesicles by Dialysis of Nonionic Alkyl Maltoside Detergents. Biochim Biophys Acta—Biomembranes 946: 379–388.
- Vanaken, T., FoxallVanaken, S., Castleman, S., and FergusonMiller, S. Alkyl Glycoside Detergents: Synthesis and Applications to the Study of Membrane Proteins. Methods Enzymol 125: 27–35.
- Saito, S. and Tsuchiya, T. 1984. Characteristics of noctyl betaDthioglucopyranoside, a nonionic detergent useful for membrane biochemistry. Biochem J 222: 829–832.
- Measurement from Affymetrix Anatrace®.
