Sep pak column

DAT was isolated from Mycobacterium fortuitum ATCC 6841; the mycobacteria were cultured for 14 days in Sauton media. Non-covalently linked lipids were extracted from the filtered biomass with CHCI₃/CH₃OH (1:2 vol/vol) for 1 h at 50°C. The solvent recovered was kept, and the filtered biomass was treated with CHCI₃/CH₃OH (2:1, vol/vol) for 1 h at 50°C. Pooled glycolipid extracts were dried and suspended in CHCl₃/CH₃OH/H₂O (4:2:1 vol/vol/vol). After that, the crude lipid extract was dissolved in chloroform and applied to a Florisil column (Biotecna Corp., Miami, FL, USA). The lipids eluted with CHCI₃/CH₃OH were monitored by thin-layer chromatography (TLC) on silica gel-60 F₂₅₄ coated plates (E. Merck, Darmstadt, Germany) and developed with CHCI₃/CH₃OH/H₂O (60:12:1, vol/vol/vol). The sugar-containing compounds were visualized by spraying the plates with 2% anthrone in concentrated H₂SO₄ followed by heating at 110°C. Acylated trehaloses appeared as anthrone-positive lipids (blue spots) with an Rf value of 0.37 for DAT. The lipids were purified with Sep-Pak columns (Waters, Milford, MA. USA) and analyzed by TLC to confirm DAT purification. The fractions with purified DAT were pooled, dried and subjected to the Lymulus test to verify endotoxin contamination (Lonza, Basel, Switzerland).

For analysis of peptides and proteins from SILAC-labeled cells, lysates were digested using LysC and trypsin whereafter the resulting peptides mixture was desalted using Sep-Pak columns (Waters). Aliquots of 2 mg of desalted peptides were separated into 46 fractions using reversed-phase chromatography with alkaline running buffers and a Ultimate 3000 high-pressure liquid chromatography (HPLC) system (Dionex) as outlined previously^{30 (link)}. Each fraction was acidified by adding formic acid to a final concentration of 0.1% and subsequently concentrated by vacuum centrifugation. The concentration of peptides was spectrophotometrically determined at 280 nm using a NanoDrop instrument (Thermo Scientific). Peptides were then diluted in 5% acetonitrile and 0.1% trifluoroacetic acid, whereafter 1 µg of peptide was loaded for LC-MS/MS analysis.
For specific analysis of the methylation status of Lys55 and the N terminus of eEF1A samples were diluted in 2 M guanidine hydrochloride, 5 mM tris(2-carboxyethyl)phosphine, 10 mM chloroacetamide, 100 mM Tris pH 8.5 and digested with with endoproteinase Glu-C (Roche) or Chymotrypsin (Roche), respectively. The resulting peptides were desalted, stored on and eluted from C₁₈ StageTips^{65 (link)}.

For each genotype, blood from four age matched adult mice was pooled to achieve a 1 ml plasma sample. Samples were concentrated on C18 columns (Sep-Pak columns; Waters), evaporated to dryness, and reconstituted in 0.9% NaCl, 0.03% acetic acid and 0.1% BSA). The levels of Ang II were quantified by RIA [26] (link), [27] (link). The results represent the arithmetic mean of assaying a minimum of 5 pools from each genotype ±95% CI for the mean. Renal Ang II was measured from age- and genotype-matched mice, as described previously [28] .

All site-specifically modified single-stranded oligonucleotides were synthesized on ABI 392 DNA synthesizer (Applied Biosystems) using standard protocol and reagents in 1 μM scale, purified by urea-PAGE, and desalted by Sep-Pak columns (Waters). Oligonucleotides containing a disulfide-bearing tether at the N⁴-position of a cytosine or at the nonbridging position of DNA backbone were synthesized and functionalized as described previously [48 (link), 49 (link)]. Oligonucleotide modification was confirmed by MALDI-TOF. All unmodified oligonucleotides were purchased from Eurofins Genomics.