Methodical analysis of protein-nitrocellulose interactions to design a refined digestion protocol Journal Article
| Authors: | Lui, M.; Tempst, P.; Erdjument-Bromage, H. |
| Article Title: | 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. |
| Keywords: | protein analysis; protein degradation; protein; immunoblotting; polyacrylamide gel electrophoresis; piperidine; cetrimide; detergent; radioiodination; digestion; acetonitrile; priority journal; article; polyoxyethylene; pyroxylin |
| Journal Title: | Analytical Biochemistry |
| Volume: | 241 |
| Issue: | 2 |
| ISSN: | 0003-2697 |
| Publisher: | Academic Press, Elsevier Inc |
| Date Published: | 1996-10-15 |
| Start Page: | 156 |
| End Page: | 166 |
| Language: | English |
| DOI: | 10.1006/abio.1996.0393 |
| PUBMED: | 8921181 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 22 November 2017 -- Source: Scopus |
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