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General Information info

Manuscript title Global analysis of protein expression in yeast.
PubMed ID 14562106
Journal Nature
Year 2003
Authors Sina Ghaemmaghami, Won-Ki Huh, Kiowa Bower, Russell W. Howson, Archana Belle, Noah Dephoure, Erin K. O'Shea & Jonathan S. Weissman
Affiliations Howard Hughes Medical Institute, University of California–San Francisco, San Francisco, California 94143-2240, USA
Keywords yeast, protein expression, S. cerevisiae
Full text article Downloadarticle Ghaemmaghami_2003.pdf
Project name not specified

Experiment Description info

Organism Saccharomyces cerevisiae
Strain TAP-tagged strain
Data type enzyme/protein concentrations
Data units molecules/cell
Execution date not specified

Experimental Details info

Temperature (0C) 30
pH not specified
Carbon source glucose,
Culture mode chemostat
Process condition aerobic
Dilution rate (h-1)
Working volume (L) 0.0017
Biomass concentration (g/L) OD of ~0.7
Medium composition

YEPD media.

General protocol information Measurement method: LC-MS/MS

Methods description - Notes

Extract preparation and quantification of protein levels: Cultures (1.7 ml) of tagged strains were grown in 96-well format to log phase, and total cell
extracts were examined by SDS–polyacrylamide gel electrophoresis (PAGE)/western blot analysis. Before the quantitative SDS–PAGE/western blot analysis, strains were ordered on the basis of estimates of TAP abundance from a preliminary dot-blot analysis. In order to provide a standard for the conversion of western signals to absolute protein levels, a TAP-tagged protein (Escherichia coli initiation factor A, INFA) was overexpressed in E. coli and purified to homogeneity. Yeast extracts containing serial dilutions of INFA ranging from 500 attomoles (which was the limit of detection, see Supplementary Fig. S1) to 25 picomoles were run on a gel along with extracts from 25 different yeast TAP-tagged strains representing the full range of observed protein signals (a second TAP-tagged protein (initiation factor B) was also analysed to ensure that the observed TAP signal was not influenced by the fusion protein). Comparison of the signals generated by these 25 proteins to the known standards allowed the creation of a conversion factor between the observed western blot signals and absolute protein levels. Based on the number of cells (1x10^7) used for the SDS–PAGE/western blot analysis, the protein levels were then converted to measurements of protein molecules per cell. Cultures were grown to OD of ~0.7 at 30 oC and pelleted cells were lysed by the addition of 50 µL of a boiling SDS solution (50mM Tris-HCl , pH 7.5, 5% SDS, 5% glycerol, 50mM DTT, 5mM EDTA, Bromophenol Blue, 2µg/mL Leupeptin, 2µg/mL Pepstatin A, 1µg/mL Chymostatin, 0.15 mg/mL Benzamidine, 0.1 mg/mL Pefabloc, 8.8 µg/mL Aprotanin, 3µg/mL Anitpain). Lysed cells were centrifuged and the supernatant extract was stored at –80 oC. 13 µL aliquots of the SDS-lysed extracts were loaded on 26 well, 4-15% gradient acrylamide Tris-HCl Criterion precast gels (Bio Rad). The gels were run at 200 Volts for 70 minutes and transferred using Trans-blot SD semi-dry transfer cell (BioRad) onto PVDF membranes at a constant current of 160 mA per gel for 120 minutes. Before transfer, the activated PVDF membranes and the gels were soaked in 39mM Glycine, 48mM Tris-HCl, 0.375% SDS with and without 20% methanol respectively in order to facilitate transfer while preventing bleed through. Analysis of a number of randomly chosen blots indicated that the large majority of the protein samples were transferred onto the PVDF membrane. The blots were probed using an affinity purified rabbit polyclonal antibody raised against the calmodulin binding peptide. This antibody can detect the TAP tag with great sensitivity as it can bind CBP as well as the Protein A segment of the tag through interaction with its Fc region. The blots were subsequently probed with a horse radish peroxidase (HRP) conjugated Goat secondary antibody (Jackson ImmunoResearch) against rabbit IgG and reacted with SuperSignal West Femto Maximum Sensitivity Substrate ECL (BioRad) and the chemiluminescence of the bands corresponding to the tagged proteins were detected and quantified using a CCD camera (Alpha Innotech). Transfer efficiency was monitored by Ponceau S staining all membranes and including “Magic Mark” (invitrogen) molecular weight standards, which contain IgG binding domains allowing visualization by Western blotting.

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Submission and curation info

Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Homepage: http://kdbio.inesc-id.pt/kimosys
Interests: mathematical modeling, accessible data, use of data

Created 2018-07-16 15:29:11 UTC

Updated 2018-07-16 15:29:11 UTC

Version 0

Status (reviewed) 2018-07-16 15:29:45 UTC




Associated Models


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Model
EntryID
Model name Category Model Type Data used for Access Json
39
Authors: Kieran Smallbone, Hanan L. Messiha, Kathleen M. Carroll, Catherine L. Winder, Naglis Malys, Warwick B. Dunn, Ettore Murabito, Neil Swainston, Joseph O. Dada, Farid Khan, Pınar Pir, Evangelos Simeonidis, Irena Spasić, Jill Wishart, Dieter Weichart, Neil W.

Original paper: A model of yeast glycolysis based on a consistent kinetic characterisation of all its enzymes.

smallbone18 Metabolism ordinary differential equations Model validation Visto4 {"affiliation":"Manchester Centre for Integrative Systems Biology, Manchester Institute of Biotechnology, The University of Manchester, UK","article_file_name":null,"authors":"Kieran Smallbone, Hanan L. Messiha, Kathleen M. Carroll, Catherine L. Winder, Naglis Malys, Warwick B. Dunn, Ettore Murabito, Neil Swainston, Joseph O. Dada, Farid Khan, P\u0131nar Pir, Evangelos Simeonidis, Irena Spasi\u0107, Jill Wishart, Dieter Weichart, Neil W.","biomodels_id":"smallbone18","category":"Metabolism","combine_archive_content_type":null,"combine_archive_file_name":null,"combine_archive_file_size":null,"combine_archive_updated_at":null,"comments":"Original model source: in JWS online database.","control":null,"dilution_rate":"","id":39,"journal":"FEBS Letters","keywords":"Glycolysis, Systems biology, Enzyme kinetic, Isoenzyme, Modelling","main_organism":"Saccharomyces cerevisiae","manuscript_title":"A model of yeast glycolysis based on a consistent kinetic characterisation of all its enzymes.","model_name":"smallbone18","model_type":"ordinary differential equations","organism_id":null,"project_name":"","pubmed_id":"23831062","review_journal_id":null,"sbml_file_name":"Smallbone_2013.pdf","software":"Copasi (www.copasi.org)","used_for":"---\n- Model validation\n","year":2013} Administrator KiMoSys



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