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Title: youngah_YAGsn2      
description:
replicate enrichment for soluble proteins; Goo et al., 2003, Mol Cell Proteomics 2(8):509 Young Ah Goo ICAT experiments - YAGsn2 directory
privacy:
not applicable
aggregation:
instance of dataset
ID:
PAe000249
refinement:
curated
availability:
available
types:
mass spectrometry
strain:
Halobacterium sp. NRC-1 (ATCC700922)
name:
Halobacterium
description:
Treatment: ; Growth: Strain Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37°C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described in Oesterhelt et al., Methods Enzymol. 31, 1974, 667–678.; Digestion: One hundred micrograms of proteins were digested with 2µg of trypsin (Promega, Madison, WI) in 50 mM sodium bicarbonate (pH 8.3) at 37°C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer’s procedure. The proteins were lyophilized and stored at -80°C and resuspended in 100µL of 0.4% acetic solution prior to mass spectrometer analysis.; Extraction: Preparation of Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (Oesterhelt et al., 1974, Methods Enzymol. 31, 667–678; DasSarma et al, 1995, Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY). One liter of Halobacterium sp. NRC-1 culture was grown to OD₆₀₀= ~2.0 and pelleted by centrifugation at 7,500 rpm at 4°C for 10 min. Pellets were resuspended in 20ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mM of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000× g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000× g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000× g at 10°C overnight. The membrane fraction was collected and washed three times in 10ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10°C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueousbasal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4°C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis.; Separation:
name:
LCQ DECA
accessURL: ftp://ftp.peptideatlas.org/pub/PeptideAtlas/Repository/PAe000249/
storedIn:
Peptide Atlas
qualifier:
gzip compressed
format:
XML
accessType:
download
authentication:
none
authorization:
none
abbreviation:
ISB
homePage: http://www.systemsbiology.org/
ID:
SCR:011305
name:
Institute for Systems Biology
homePage: http://www.peptideatlas.org/
ID:
SCR:006783
name:
PeptideAtlas

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