Chapter title |
Enrichment of Low-Abundant Protein Targets by Immunoprecipitation Upstream of Mass Spectrometry
|
---|---|
Chapter number | 12 |
Book title |
Proteomic Profiling
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2550-6_12 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2549-0, 978-1-4939-2550-6
|
Authors |
Barbara Kaboord, Suzanne Smith, Bhavin Patel, Scott Meier |
Abstract |
Immunoprecipitation (IP) is commonly used upstream of mass spectrometry (MS) as an enrichment tool for low-abundant protein targets. However, several aspects of the classical IP procedure such as nonspecific protein binding to the isolation matrix, detergents or high salt concentrations in wash and elution buffers, and antibody chain contamination in elution fractions render it incompatible with downstream mass spectrometry analysis. Here, we discuss two IP workflows that are designed to minimize or eliminate these contaminants: the first employs biotinylated antibodies and streptavidin magnetic beads while the second method utilizes a traditional antibody that is oriented and cross-linked to Protein AG magnetic beads. Both modified magnetic supports have low background binding and both antibody immobilization strategies significantly reduce or eliminate antibody heavy and light chain contamination in the eluent, minimizing potential ion suppression effects and thereby maximizing detection of target antigens and interacting proteins. |
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