Ted by means of a microbiological inoculation loop. Seventeen further fractions of 800 l each have been taken having a pipette tip from the leading to bottom with the tube. For protein identification by mass spectrometry (MS), proteins have been separated by polyacrylamide gels (Novex NuPAGE 4 to 12 Bis-Tris gel). Lanes had been cut into 22 equally spaced pieces with an in-house created gelcutter. The sample was digested with trypsin (sequencing grade-modified trypsin; Promega) as described HSV-1 Inhibitor site previously (26), and peptides have been analyzed subsequently on a hybrid triple quadrupole/linear ion-trap mass spectrometer (4000 QTRAP; Applied Biosystems/MDS Sciex) coupled to a one-dimension (1D) nano-liquid chromatography (LC) technique (Eksigent). 5 microliters (ten sample) was CXCR7 Activator Accession injected onto a PepMap RPC18 trap column (300- m inside diameter [i.d.] by five mm; 5- m particle size; C18 column with 100-?pore size [Dionex]), purified, and desalted with 0.1 (vol/vol) formic acid? (vol/vol) CH3CN at 30 l/min (all Biosolve). Samples have been separated by gradient elution onto a PepMap C18 microcolumn (75- m i.d. by 15 cm; 3- m particle size; C18 column with 100-?pore size [Dionex]) using a linear gradient of 2 to 45 (vol/vol) CH3CN?0.1 (vol/vol) formic acid at 250 nl/min. Analyst, version 1.four.1, and Bioanalyst, version 1.four.1, application applications (Applied Biosystems/MDS Sciex) have been applied for acquisition control. Tandem MS (MS/MS) spectra have been searched against a nonredundant sequence database at www .dictybase.org (27) utilizing MASCOT (version 2.two.05; Matrix Science). Tolerances for peptides have been set to 1.5 Da and 0.five Da for MS and MS/MS, respectively. Identified proteins have been accepted using a minimum total score of 50 and at least two distinctive peptides using a minimum peptide score of ten. Western blotting employed the PDI antibody or antibodies recognizing GFP MAb 264-449-2 (offered from Millipore), mitochondrial porin MAb 70-100-1 (28), severin MAb 42-65-11 (29), and FcsA MAb 221457-5 (15). The work by von L neysen et al. (15) also describes how the mode of membrane association was determined by differential centrifugation, extraction, and subsequent Western blotting. Lipid analysis. To establish the TAG content of a whole-cell homogenate enzymatically, about 2.5 107 washed cells had been resuspended in 200 l of thin-layer chromatography (TLC) buffer, frozen in liquid nitrogen, and thawed at 37 3 times in order that cells have been disrupted andec.asm.orgEukaryotic CellLipid Droplets in Dictyosteliumcellular lipids have been released. A sample of 50 l of the sample was added to 1 ml of TAG reagent (LT-SYS, Berlin, Germany) and incubated for 20 min at room temperature inside a cuvette in the dark. This leads to the release of glycerol from fat, a phosphorylated intermediate, and its subsequent conversion to dihydroxyacetone phosphate and hydrogen peroxide. The latter metabolite is photometrically detected as the formation of quinoneimine, absorbing at 500 nm. For lipid analysis by thin-layer chromatography (TLC), the classical strategy of Bligh and Dyer (30) was adapted as follows. About five 107 washed cells have been resuspended in 1 ml of TLC buffer (20 mM HEPES, 150 mM NaCl, pH 7.5), and an suitable aliquot (in accordance with the previously determined protein content by the bicinchoninic acid (BCA) strategy, per the manufacturer’s instructions [Pierce]) was adjusted to 1.two ml with TLC buffer. First, 4.five ml of 1:two chloroform-methanol was added and mixed for 1 min. Subsequent, 1.5 ml of chloroform and finally 1.5 ml of doubl.