کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2022112 1069278 2006 13 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Capturing protein–protein complexes at equilibrium: The holdup comparative chromatographic retention assay
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Capturing protein–protein complexes at equilibrium: The holdup comparative chromatographic retention assay
چکیده انگلیسی

The popular pulldown chromatographic assay detects complexes mediated by fusion proteins retained on affinity resin. The main limitation of this method is that it does not analyze complexes at equilibrium but after several washing steps. Consequently, fast-dissociating complexes may remain undetected. Here, we present the holdup assay, based on the principle of comparative chromatographic retention which eliminates the use of washing steps. The assay evaluates fractions of free and bound species at equilibrium. We used human papillomavirus oncoprotein E6, an E6-binding peptide and an E6-binding PDZ domain, to test several protocols utilizing pure proteins or expression extracts. The holdup assay is faster and more informative than the pulldown assay. It detects fast-dissociating complexes and it is also suited for evaluating equilibrium constants. It is potentially adaptable for automated determination of affinity constants and high-throughput analysis of interactions between proteins and other proteins, peptides, nucleic acids, or small regulatory molecules.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 50, Issue 1, November 2006, Pages 89–101
نویسندگان
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