Journal: The Journal of biological chemistry

Article Title: Lipid dependence and basic kinetics of the purified 1,2-diacylglycerol 3-glucosyltransferase from membranes of Acholeplasma laidlawii.

doi: 10.1074/jbc.272.2.929

Figure Lengend Snippet: FIG. 1. Purification of the 1,2-diacylglycerol 3-glucosyltrans- ferase in three column chromatography steps. Protein (E) and MGlcDAG synthesis activity (G) were eluted in three different column chromatography steps as described under “Materials and Methods.” A, IEC on SP-Sepharose (Pharmacia), pH 8. The solid black line is the applied NaCl concentration. B, gel filtration on Sephacryl S-100 HR (Pharmacia); one of the four best fractions from the former IEC step. C, HAC on ceramic hydroxyapatite Macro-Prep (Bio-Rad), pH 8. The solid black line is the applied KH2PO4/K2HPO4 concentration. The MGlcDAG synthesis activity was assayed as described under “Materials and Meth- ods” with 27 mM amphiphiles present (3.7 mol % DOG, 33 mol % DOPG, and 63 mol % CHAPS).

Article Snippet: Hydroxyapatite Chromatography (HAC)—A column (16 mm in diameter) packed with 6 ml of ceramic hydroxyapatite Macro-Prep (Bio-Rad) was equilibrated with HAC-buffer: 100 mM KH2PO4/K2HPO4, pH 8, 20% (v/v) glycerol, and 20 mM CHAPS.

Techniques: Purification, Column Chromatography, Activity Assay, Concentration Assay, Filtration

Journal:

Article Title: Purification, cloning and characterization of a GPI inositol deacylase from Trypanosoma brucei

doi: 10.1093/emboj/20.17.4923

Figure Lengend Snippet: Fig. 3. Purification and microsequencing of the inositol deacylase. [3H]DFP-labelled bloodstream-form T.brucei membranes (4 × 1011 cell equivalents) were washed with high-pH buffer and extracted with nOG. The soluble fraction (lane 1) was adsorbed with anti-VSG–Sepharose and the supernatant (lane 2) was adsorbed with ConA–agarose. The unbound material (lane 3) was discarded and the α-methyl-mannoside eluate (lane 4) was fractionated by TMAE–Fractogel FPLC. The radioactive fractions eluting at 400 mM NaCl (lane 5) were diafiltered and applied to a hydroxyapatite FPLC column. An aliquot (1%) of the radioactive fraction eluting at 200 mM sodium phosphate (lane 6) was analysed by SDS–PAGE and fluorography (lane 7). The silver-stained gel (lanes 1–6) represents 0.0005% (lanes 1–3) or 1% (lanes 4–6) of the total material. A preparative gel of the material shown in lanes 6 and 7 was used to excise the band indicated by the arrow for in-gel trypsin digestion. The tryptic peptides were fractionated by capillary microbore HPLC and the Edman sequencing results of five peptides are indicated. Residues in parentheses were ambiguous.

Article Snippet: The sample was applied to a ceramic hydroxyapatite FPLC column (CHT2-1; Bio-Rad), washed with 6 ml starting buffer and eluted with a gradient to 500 mM sodium phosphate pH 7.2, 0.1% nOG over 30 min at 1 ml/min.

Techniques: Purification, SDS Page, Staining, Sequencing

Journal: bioRxiv

Article Title: A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis

doi: 10.1101/691352

Figure Lengend Snippet: LC-MS/MS analysis of chemically phosphorylated synthetic peptides after alkaline pHis enrichment. A . Three liquid-chromatography profiles represent the pool of synthetic peptides (NME1, PGAM1, histone H4) chemically phosphorylated with no enrichment, after 1/3-pHis ImmunoAffinity Purification (IAP) alone, or successive HAP+IAP, from top to bottom respectively. HAP for Hydroxyapatite. B . MS profile from combined HAP+IAP enrichment (retention time window: 10-28 min). Peptide sequences are indicated through color blue (histone H4), red (NME1) and green (PGAM1), and the charge state (z) is symbolized by a square (+1), triangle (+2) or a star (+3). Peaks corresponding to the phosphorylation forms are manually annotated with P or PP. C . Theoretical monoisotopic mass/charge (m/z) ratio for each non-phosphorylated and phosphorylated peptide sequences used for manual annotations. (using MS-product from UCSF - Protein Prospector V5.20.0 - Baker, P.R. and Clauser, R. http://prospector.ucsf.edu ).

Article Snippet: Chromatography column were prepared using HAP resin (Bio-Rad, 1300420) or SAX resin (Poros 50HQ, #82077 Thermo scientific).

Techniques: Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Immunoaffinity Purification

Journal: bioRxiv

Article Title: A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis

doi: 10.1101/691352

Figure Lengend Snippet: Proportion of phosphoresidues after phosphoenrichment. Values indicated correspond to the number of phosphorylation sites identified by MS/MS and their percentage per residue. Only O-phosphate (STY) and N-phosphate (HKR) residues were integrated in MaxQuant analysis for each enrichment method, considering the main neutral loss of 98 Da for the N-phosphate residue. Only phosphorylation sites identified with a probability of localization >0.75 are shown. No phosphorylation in C-ter position were considered. A. and B . Global phosphopeptide enrichment from HAP (hydroxyapatite) or SAX (strong anion-exchange) chromatography using the same lysate sample. C- Unique IAP enrichment using 6 combined pHis mAbs (1-pHis clones: SC1-1, SC50-3, SC77-11; 3-pHis clones: SC39-6, SC44-1, SC56-2). D. and E . Combination of global phosphopeptide enrichment, followed by 1/3-pHis immunoaffinity purification using the same lysate sample.

Article Snippet: Chromatography column were prepared using HAP resin (Bio-Rad, 1300420) or SAX resin (Poros 50HQ, #82077 Thermo scientific).

Techniques: Tandem Mass Spectroscopy, Chromatography, Clone Assay, Immunoaffinity Purification

Journal: bioRxiv

Article Title: A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis

doi: 10.1101/691352

Figure Lengend Snippet: Numbers of each phosphoresidue localized with a high probability within a peptides with 1 to 5 phosphates. A. and B . Global phosphopeptide enrichment from HAP or SAX chromatography. C . Unique IAP enrichment using 6 combined pHis mAbs (1-pHis clones: SC1-1, SC50-3, SC77-11; 3-pHis clones: SC39-6, SC44-1, SC56-2). D. and E . Combination of global phosphopeptides enrichment followed by 1/3-pHis immunoaffinity purification

Article Snippet: Chromatography column were prepared using HAP resin (Bio-Rad, 1300420) or SAX resin (Poros 50HQ, #82077 Thermo scientific).

Techniques: Chromatography, Clone Assay, Immunoaffinity Purification