Methodical analysis of protein-nitrocellulose interactions to design a refined digestion protocol.
We have analyzed the efficacies of seven different organic solvents, 13 organic bases, and 17 detergents to dissociate electroblotted proteins from nitrocellulose. Most efficient were a 1% piperidine-40% acetonitrile mixture and 1% concentrations of either cetyltrimethylammonium bromide or several polyoxyethylene and zwitterionic detergents at pH > or = 8.5. In general, detergent-promoted elution varied with pH (8.5-->12.0) and temperature (37-->65 degrees C) in a detergent-dependent and protein-dependent manner, making for unpredictable results of any combination. However, Zwittergent 3-16, as the major exception, eluted proteins of different sizes and properties almost equally well at pH 8.5. This preferred effect is not enhanced by addition of organic solvent. In fact, detergent/acetonitrile mixtures were generally less efficient for protein elution. Zwittergent 3-16 (1% in 100 mM NH4HCO3) proved to be the most powerful additive for one-step enzymatic digestion of nitrocellulose-bound proteins, both in terms of highest recoveries (including endoproteinase Lys-C digestions) and general applicability, a major improvement over the use of Tween 80 and hydrogenated Triton X-100. The new digest procedure takes just 2 h to complete and requires 5 ng protease per 1 microliter buffer per 1 mm2 of nitrocellulose; relatively large pieces of membrane (> or = 100 mm2) can be successfully processed. The same detergent is a satisfactory additive for digestion of polyvinylidene difluoridebound proteins as well. Moreover, Zwittergent 3-16 is fully compatible with the usual downstream procedures. It is uv (OD214) transparent and does not interfere with high-performance liquid chromatography in any other way. Apparent incompatibility with direct (i.e., "prechromatography") matrix-assisted laser-desorption ionization time-of-flight mass spectrometry can be either reduced by methanol washing of the dried sample and activation of a low-mass gate or eliminated by a simple microliter cleanup procedure.
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Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. p-tempst@mskcc.org
Tempst
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Erdjument-Bromage
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D002851:Chromatography, High Pressure Liquid; D003101:Collodion / Q000737:chemistry*; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008722:Methods; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry*; D012997:Solvents; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D005465:Fluorocarbon Polymers
D003101:Collodion; D003902:Detergents; D011145:Polyvinyls; D011506:Proteins; D012997:Solvents; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers