title: A new high-performance thin layer chromatography-based assay of detergents and surfactants commonly used in membrane protein studies.
abstract: The hydrophobic nature of membrane proteins (MPs) necessitates the use of detergents for their extraction, solubilization and purification. Because the concentration of amphiphiles is crucial in the crystallization process, detergent quantification is essential to routine analysis. Here we describe a quantitative high-performance thin-layer chromatography (HPTLC) method we developed for the detection of small quantities of detergent bound to solubilized MPs. After optimization of aqueous deposit conditions, we show that most detergents widely used in membrane protein crystallography display distinctive mobilities in a mixture of dichloromethane, methanol and acetic acid 32:7.6:0.4 (v/v/v). Migration and derivatization conditions were optimized with n-dodecyl-β-D-maltoside (DDM), the most popular detergent for membrane protein crystallization. A linear calibration curve very well fits our data from 0.1 to 1.6 μg of DDM in water with a limit of detection of 0.05 μg. This limit of detection is the best achieved to date for a routine detergent assay, being not modified by the addition of NaCl, commonly used in protein buffers. With these chromatographic conditions, no prior treatment is required to assess the quantities of detergent bound to purified MPs, thus enabling the quantification of close structure detergents via a single procedure. This HPTLC method, which is fast and requires low sample volume, is fully suitable for routine measurements.
authors: CEA, IBEB, Lab Bioenerget Cellulaire, Saint-Paul-lez-Durance, F-13108, France.
authors: Laurie-Anne
authors: LA
authors: Barret
authors: Ange
authors: A
authors: Polidori
authors: Françoise
authors: F
authors: Bonneté
authors: Pierre
authors: P
authors: Bernard-Savary
authors: Colette
authors: C
authors: Jungas
mesh_terms: D002138:Calibration; D003585:Cell Biology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer / Q000379:methods*; D003902:Detergents / Q000032:analysis* / Q000737:chemistry; D005960:Glucosides / Q000032:analysis / Q000737:chemistry; D057230:Limit of Detection; D009994:Osmolar Concentration; D013501:Surface-Active Agents / Q000032:analysis* / Q000737:chemistry
chemical_list: D003902:Detergents; D005960:Glucosides; D013501:Surface-Active Agents; C040358:dodecyl maltoside