Methodical analysis of protein-nitrocellulose interactions to design a refined digestion protocol

Abstract

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 ≤ 8.5. In general, detergent-promoted elution varied with pH (8.5 → 12.0) and temperature (37 → 65°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 μl buffer per 1 mm2 of nitrocellulose; relatively large pieces of membrane (≤ 100 mm2) can be successfully processed. The same detergent is a satisfactory additive for digestion of polyvinylidene difluoride-bound 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.