(like SDS, n-octyl-β-d-glucopyranoside (OG)
, n-dodecyl-β-d-maltoside (DDM)
are widely used in membrane protein solubilization even though it is well known that different detergents have different weaknesses. Short chain nonionic detergents for example can affect the functional properties of a membrane protein (1). It seems clear that removing the native lipid bilayer from the membrane protein can interfere with the function of the protein. One way to mimic the native lipid membrane are MSP-nanodiscs
(Fig. 1) or detergent-free polymer systems (Fig. 2) (Styrene-maleic acid co-polymers (SMAs) (2), Diisobutylene-maleic acid (DIBMA)) (3,4). With the latter you can directly extract membrane proteins from cells without an intermediate step of detergent solubilization (5). Synthetic polymers have to carry a styrene or maleic acid group himself to solubilize proteins.
Fig.1: Nanodisc with a target protein and membrane scaffold proteins (MSPs, green).
Fig.2: Synthetic Polymers like SMA or DIBMA carrying a target protein.
Why it is advantageous to use PureCube DIBMA and not SMAs for your protein solubilization?
SMAs have big advantages in contrast to many detergents and are succsessfully used for many applications (6). The drawback of SMAs is a high absorbance of ultraviolet light in solution with an absorbance maxima at 280 nm. The main reason for this peak at 280 mn are aromatic amino acids like tryptophan or phenylalanine, SMA itself carries an aromatic ring. So quantitative protein concentration measuring of your sample is not possible during the process of membrane protein solubilization, stabilization and purification. With PureCube DIBMA from Cube Biotech you can solubilize, stabilize and purify your protein detergent-free and measure your protein concentration without any problems.
Fig.3: SDS Page showing solubilized membrane-protein fractions of E. coli. 10 mM (0.5 % (w/v)) DDM and 3 mM (2.5 % (w/v)) DIBMA was used. The figure is taken from Grethen et. al. 2017 (7).
Fig.4: Chemical structural formula of diisobutylene-maleic acid (DIBMA).
Why you should use PureCube DIBMA from Cube Biotech?
We provide our product highly purified and lyophilized. On top of that, PureCube DIBMA is lyophilized from two different buffer solutions (HEPES or TRIS) to ensure a stable pH at 7.5 which is ideal for most protein solubilizations. Feel free to contact
us if you wish to have PureCube DIBMA in a different kind of buffer composition. For different applications you can choose from samples with a medium length of 10,000 or 12,000 g/mol.
Fig. 5: DIBMA solubilizes protein from Pellet, supernatant 9000 xg and pellet 100000 xg. Differernt concentrations of DIBMA were used to determine the perfect solubilization conditions.
||~12,000 g/mol or 10,000 g/mol
||> 10 % in H2O
|Absorbance at 280 nm
||< 0.3 (1 % solution)
|A.||Protocol for solubilization of membrane proteins using DIBMA:|
- With DIBMA you can solubilize membrane proteins in:
You can add directly the isolated membrane protein solution (40 mg/ml (w/v)) (NOTE: pH is already adjusted with TRIS or HEPES) to your DIBMA/Buffer powder to a final concentration of 0,4 % - 4 %. We advise you to add 2 ml of your solution to 50 mg lyophilized DIBMA to start with a DIBMA concentration of 2,5 %. For screening purposes you can try different DIBMA concentrations to find optimized conditions for solubilization.
- Suspensions of whole cells
- Supernatant 9,000
- Homogenized 100,000 Pellet
Optional: It is possible to screen the total protein concentration (beginning from 40 mg/ml) against 2,5 % DIBMA. The DIBMA or protein concentration screening can influence the success of the solubilization of your target protein.
With gentle shaking overnight at 4 °C and/or room temperature you can solubilize your proteins. The optimal conditions are protein specific (screen!).
The contained TRIS/HEPES buffer keeps the pH at 7.5 which stabilize the functionality of DIBMA and is suitable for the most applications. If you need a different pH or buffer for your protein solubilization we provide DIBMA without buffer. Please note that a pH smaller than 6.5 is not suitable for solubilization with DIBMA.
| B.||SDS-PAGE Protocol:|
If you want to run a SDS Page your proteins have to be separated from the synthetic polymers. The presence of the polymers can lead to smearing bands on SDS-PAGE gels (8,9). Wessel and Flügge discribed the protocol in 1984 originally (10). It was modified in 2017 for the use with DIBMA (8).
- Measure volume of solubilized protein - polymer sample
- Vortex the sample with 4x volume of cold methanol
- Vortex with 1x volume of chloroform
- Centrifuge for 3 min at 4 °C and 15,000 g with 3x volume of cold water
- Add 4x volume of methanol to the organic layer and discard the aqueous.
- Centrifuge at 20,000 g for 5 min at 4 °C.
Frequently asked questions
|Is DIBMA from Cube Biotech ready to use?
|Yes, our DIBMA is ready to use. You can start directly with solubilization. Read our protocol for more information.
Which pH is suitable for DIBMA?
|For DIBMA a pH of 7.5 is recommended. DIBMA does not solubilize if the pH is smaller than 6.5.
Which concentrations of DIBMA should I use for my protein?
|In general we advise you to add 2,5 % DIBMA to your solution. But the optimal conditions have to be screened by yourself (Fig.5).
I used DIBMA for protein solubilization and a white precipitate appeared - what happened?
|Your DIBMA precipitated. You should check your pH and ensure that your pH never drops down to 6.5.
- Seddon, Annela M., Paul Curnow, and Paula J. Booth. "Membrane proteins, lipids and detergents: not just a soap opera." Biochimica et Biophysica Acta (BBA)-Biomembranes 1666.1-2 (2004): 105-117.
- Knowles, Timothy J., et al. "Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer." Journal of the American Chemical Society 131.22 (2009): 7484-7485.
- Oluwole, Abraham Olusegun, et al. "Solubilization of Membrane Proteins into Functional Lipid‐Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer." Angewandte Chemie International Edition 56.7 (2017): 1919-1924.
- Oluwole, Abraham Olusegun, et al. "Formation of lipid-bilayer nanodiscs by diisobutylene/maleic acid (DIBMA) copolymer." 33.50 (2017): 14378-14388.
- Long AR, O’Brien CC, Malhotra K, Schwall CT, Albert AD, Watts A, Alder NN (2013) A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs. BMC Biotechnol 13:41
- Dörr, Jonas M., et al. "The styrene–maleic acid copolymer: a versatile tool in membrane research." European Biophysics Journal 45.1 (2016): 3-21.
- Grethen, Anne, et al. "Thermodynamics of nanodisc formation mediated by styrene/maleic acid (2: 1) copolymer." Scientific reports 7.1 (2017): 11517.
- Oluwole, Abraham Olusegun, et al. „Solubilization of Membrane Proteins into Functional Lipid‐Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer.“ Angewandte Chemie International Edition 56.7 (2017): 1919-1924.
- Lee, Sarah C., et al. „A method for detergent-free isolation of membrane proteins in their local lipid environment.“ Nature protocols 11.7 (2016): 1149.
- Wessel, D. M., and U. I. Flügge. „A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.“ Analytical biochemistry 138.1 (1984): 141-143.