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Beads-free protein immunoprecipitation for a mass spectrometry-based interactome and posttranslational modifications analysis

BACKGROUND: Protein immunoprecipitation (IP) coupled with MS provides means to interrogate protein complexes and their posttranslational modifications (PTMs). In a typical protein IP assay antibodies are conjugated to protein A/G beads requiring large amounts of antibodies, tube transfers and centri...

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Detalles Bibliográficos
Autores principales: Mikula, Michal, Rubel, Tymon, Karczmarski, Jakub, Statkiewicz, Malgorzata, Bomsztyk, Karol, Ostrowski, Jerzy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4557753/
https://www.ncbi.nlm.nih.gov/pubmed/26336360
http://dx.doi.org/10.1186/s12953-015-0079-0
Descripción
Sumario:BACKGROUND: Protein immunoprecipitation (IP) coupled with MS provides means to interrogate protein complexes and their posttranslational modifications (PTMs). In a typical protein IP assay antibodies are conjugated to protein A/G beads requiring large amounts of antibodies, tube transfers and centrifugations. RESULTS: As an alternative, we present Matrix-IP, beads-free microplate-based platform with surface-immobilized antibodies. Assay utilizes standard 96-well polypropylene PCR plates that are laboratory-fabricated with UV-C light and then protein A/G coated prior to IP reaction. We demonstrate application of Matrix-IP platform in MS analysis of heterogeneous nuclear ribonucleoprotein K (hnRNP K) interactome and PTMs. CONCLUSION: Matrix-IP is time-saving, easy to use high throughput method adaptable for low sample amounts and automation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12953-015-0079-0) contains supplementary material, which is available to authorized users.