A new high-performance thin layer chromatography-based assay of detergents and surfactants commonly used in membrane protein studies

Barret, Laurie-Anne; Polidori, Ange; Bonneté, Françoise; Bernard-Savary, Pierre; Jungas, Colette

doi:10.1016/j.chroma.2013.01.061

Cited by 9 publications

(5 citation statements)

References 22 publications

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“…The nature and amount of phospholipids bound to RC-LH1-PufX have been characterized using a new high performance thin layer chromatography (HPTLC) assay. 47 It seems (Barret et al, personal communication) that when purified in PCCαM, RC-LH1-PufX associates 25% more phospholipids than when purified in DDβM. This result has to be correlated with the nondetergent effect of PCCαM and its higher hydrophobicity.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…It has recently been reported that adding phospholipids, which stabilizes MP structures, leads to MP crystals that diffract to better resolution. The nature and amount of phospholipids bound to RC-LH1-PufX have been characterized using a new high performance thin layer chromatography (HPTLC) assay . It seems (Barret et al, personal communication) that when purified in PCCαM, RC-LH1-PufX associates 25% more phospholipids than when purified in DDβM.…”

Section: Resultsmentioning

confidence: 99%

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Influence of Hydrophobic Micelle Structure on Crystallization of the Photosynthetic RC-LH1-PufX Complex from Rhodobacter blasticus

et al. 2013

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Small angle X-ray scattering (SAXS) experiments are performed on two non-ionic surfactants, the dodecyl β-maltoside (DDβM) and the propyl(bi)cyclohexyl α-maltoside (PCCαM), a maltoside derivative containing a rigid bicyclohexyl group as hydrophobic chain, in order to compare the influence of both hydrophobic moiety structure and anomeric form on micelle form factors and intermicellar interactions relevant for membrane protein crystallization. Density and refractive index measurements were performed in order to determine volumetric and optical properties of surfactants, essential for determination of micelle molar masses by both SAXS and SEC-MALLS. SAXS form factors were analyzed by Guinier approximation and inverse Fourier transformation, to obtain the radius of gyration (RG) and the pair distribution function (P(r)) of each surfactant. Form factor model fitting was also performed to describe the shape and the assembly of both surfactant micelles. Finally, second virial coefficients were measured at different percentages of polyethylene glycol 3350, in order to correlate surfactant intermicellar interactions and RC-LH1-PufX phase diagram. It is thus found that while size, shape, and dimensions of micelles are slightly similar for both surfactants, their molar mass and aggregation number differ significantly. PCCαM are more densely packed than DDβM, which reflects (1) an increase in van der Waals contacts between PCCαM hydrophobic chains in the micelle bulk and (2) a supplementary intermicellar attraction compared to DDβM. Finally addition of PEG, which induces a depletion attraction, decreases the solubility of the RC-LH1-PufX complex in PCCαM.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Influence of Hydrophobic Micelle Structure on Crystallization of the Photosynthetic RC-LH1-PufX Complex from Rhodobacter blasticus

et al. 2013

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“…However, only a few types of radioactive detergents are available, making this approach generally untenable. Other methods developed to measure detergent concentrations include: (i) colorimetric assays to estimate the sugar moiety for specific detergents 5 6 ; (ii) Fourier transform Infrared spectroscopy 7 ; (iii) plain thin layer chromatography coupled with densitometric quantification, or more recently coupled with laser densitometry 8 9 ; drop-shape based quantification 10 ; (iv) liquid chromatography/ESI-MS 11 ; (v) size-exclusion chromatography coupled with multi-angle laser light scattering 12 13 and analytical ultracentrifugation 14 . Although useful, these methods are laborious, difficult to implement routinely, limited to a given type of detergent or inapplicable to detergent mixtures.…”

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confidence: 99%

Quantification of Detergents Complexed with Membrane Proteins

Chaptal¹,

Delolme²,

Kilburg³

et al. 2017

Sci Rep

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Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.

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“…Absorbance at 275 and 280 nm can be used to estimate the concentration of Triton X agent (100 and 114) and NP-408; however, the limitation of this method is that it requires subtraction of protein contribution to the signal, and this contribution is impossible to pre-estimate in complex biological samples (Antharavally et al, 2011). High-performance thin-layer chromatography has been used to analyze a mixture containing up to ten different detergents; however, the method requires that these detergents exhibit differential mobility in the liquid phase, and therefore it is not completely universal (Barret et al, 2013). Other general methods for detergent quantitation include the use of mass spectrometry (Antharavally et al, 2011) and refractive index (Strop and Brunger, 2005).…”

Section: Commentary Background Informationmentioning

confidence: 99%

Detergent Analysis in Protein Samples Using Mid‐Infrared (MIR) Spectroscopy

Das¹,

Nadler²,

Strug³

2015

CP Protein Science

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Quantitating relative levels of detergent present in protein preparations or samples derived from biological material, such as tissue or body fluids, is important because the presence of detergent may affect downstream analyses as well as protein structure/function. Especially because sample volumes, analysts' available time, and other resources may be limited, a method that consumes little sample and that is rapid and simple is needed for detergent analysis. It would also be preferable to have a method that is generally applicable across many aliphatic chain-containing molecules with many different physical properties. In this unit, methods are described for analyzing detergents and proteins in detergent-protein mixtures using mid-infrared (MIR) spectroscopy. A protocol is also included for efficient removal of unbound detergents from a protein sample accompanied by MIR-based monitoring of both detergent and protein content. This rapid monitoring of sample preparation during the workflow enables users to make timely decisions about sample preparation strategies that maximize both analyte purity and yield.

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A new high-performance thin layer chromatography-based assay of detergents and surfactants commonly used in membrane protein studies

Cited by 9 publications

References 22 publications

Influence of Hydrophobic Micelle Structure on Crystallization of the Photosynthetic RC-LH1-PufX Complex from Rhodobacter blasticus

Influence of Hydrophobic Micelle Structure on Crystallization of the Photosynthetic RC-LH1-PufX Complex from Rhodobacter blasticus

Quantification of Detergents Complexed with Membrane Proteins

Detergent Analysis in Protein Samples Using Mid‐Infrared (MIR) Spectroscopy

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