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.
||~12,000 g/mol or 10,000 g/mol
||> 10 % in H2O
|Absorbance at 280 nm
||< 0.3 (1 % solution)
||Dependend on DIBMA product
Increased with less charged DIMBAs
||Dependend on DIBMA product
Increased with less charged DIMBAs
We have written a comprehensive guide regarding nanodiscs of all kinds.
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 successfully used for many applications (6). The drawback of SMAs is a high absorbance of ultraviolet light in solution with an absorbance maximum at 280 nm. The main reason for this peak at 280 nm 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.
Less charged DIBMA
One challenge when working with traditional DIBMA is its sensitivity to the presence of ions inside the buffer / media. The reason for this is DIBMAs own charge that lets it interact with these ions. This leads to precipitation of DIBMA and therefore loss of its function. To overcome this issue we modified DIBMA to have a reduced charge to drastically reduce the affinity to the ions. This was achieved by adding either Glucosamine or an amino-functionalized diol to DIBMA (see figure 6 and 7). These two modified DIBMAs can be used for experiments for which the presence of ions is crucial for their success.
Fig. 6: Chemical structural formula of diisobutylene-maleic acid (DIBMA) Glucosamine.
Fig. 7: Chemical structural formula of diisobutylene-maleic acid (DIBMA) Glycerol.
As figure 8 shows the increased tolerance of Ca2+
is necessary. Normally DIBMA starts to precipitate in Ca2+
holding buffers at concentration of around 25 mM. Using DIBMA-Glycerol this tolerance increases to 50 mM. There is no precipitate visible at the bottom of the tube. In comparison normal DIBMA shows a visible precipitate at 25 mM. In terms of Mg2+
tolerance traditional DIBMA and the Glycerol-DIBMA both show a high tolerance above 50 mM.
Fig. 8: DIBMA precipitation in relation to the ion concentration. The shown concentrations start at 5 mM and are increasing in 5 mM steps up to 50 mM.
Our DIBMA product assortment:
In some cases the charge of normal DIBMA leads to complications when solubilizing and stabilizing certain proteins.
The reasons for this vary from protein to protein. To overcome all possible issues at once, we developped Glucosamin and Glycerol DIBMA. Through coupling on of these groups to DIBMA its own charge is reduced and nor charge- or ion related complications occur anymore.
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.