Document Detail


A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility.
MedLine Citation:
PMID:  21964698     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract, similar protein amounts as with full media are obtained, without compromising on isotope incorporation. In single and dual amino acid labeling experiments incorporation ratios are consistently ≥90% for all amino acids tested. This enables NMR studies of eukaryotic proteins and their interactions even for proteins with low expression levels. We show applications with human kinases, where protein-ligand interactions are characterized by 2D [(15)N, (1)H]- and [(13)C, (1)H]-HSQC spectra.
Authors:
Alvar D Gossert; Alexandra Hinniger; Sascha Gutmann; Wolfgang Jahnke; André Strauss; César Fernández
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Publication Detail:
Type:  Journal Article     Date:  2011-10-02
Journal Detail:
Title:  Journal of biomolecular NMR     Volume:  51     ISSN:  1573-5001     ISO Abbreviation:  J. Biomol. NMR     Publication Date:  2011 Dec 
Date Detail:
Created Date:  2011-11-23     Completed Date:  2012-03-12     Revised Date:  2012-06-22    
Medline Journal Info:
Nlm Unique ID:  9110829     Medline TA:  J Biomol NMR     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  449-56     Citation Subset:  IM    
Affiliation:
Novartis Institutes for BioMedical Research, Basel, Switzerland. alvar.gossert@novartis.com
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MeSH Terms
Descriptor/Qualifier:
Amino Acids / analysis,  chemistry,  metabolism
Animals
Baculoviridae / chemistry
Cells, Cultured
Humans
Insects
Isotope Labeling / methods*
Nuclear Magnetic Resonance, Biomolecular / methods
Proto-Oncogene Proteins c-akt / analysis*,  chemistry*
Chemical
Reg. No./Substance:
0/Amino Acids; EC 2.7.11.1/AKT2 protein, human; EC 2.7.11.1/Proto-Oncogene Proteins c-akt

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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