100 μg of protein from each brain sample was dried down and resuspended in 100 μL 8M urea/0.4 M ammonium bicarbonate for reduction and alkylation with a standard protocol [20 ] followed by overnight trypsin digestion at 37°C. The samples were cleaned using Oasis HLB 200 mg extraction cartridges (Waters) as reported previously. [21 (link)] The samples were dried by vacuum centrifugation and resuspended with the dissolution buffer from the 8-plex iTRAQ kit (AB Sciex). The manufacturer’s suggestions were followed for labeling each sample with a different iTRAQ reagent (Figure 1 ). The labeled peptides were pooled and diluted to 20 mL with 0.1% (v/v) trifluoroacetic acid (TFA). Oasis HLB 500 mg extraction cartridges (Waters) were utilized with the same protocol for clean-up.
Oasis hlb 200 mg extraction cartridges
Manufactured by Waters Corporation
Sourced in Ireland
The Oasis HLB 200 mg extraction cartridges are a laboratory equipment product designed for solid-phase extraction (SPE) applications. The cartridges feature a hydrophilic-lipophilic balanced (HLB) sorbent material that can be used to extract and purify a variety of analytes from liquid samples.
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Lab products found in correlation
2 protocols using oasis hlb 200 mg extraction cartridges
1
Quantitative Proteomics of Brain Samples
2
Tetracycline Quantification by LC-MS/MS
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To accurately measure the TC concentrations within the aquaria a LC-MS/MS method was developed. A full description of the method and validation of the performance can be found in Appendix A. Briefly, 10 mL of sample were mixed with 4 mL extraction buffer (50 mM acetic acid and 10 mM EDTA in MilliQ water, pH 3.5) and 300 ng of labeled internal standard, tetracycline-D6 (Toronto Research Chemicals, Canada), was added to the sample. Solid phase extraction was performed using Oasis HLB, 200 mg extraction cartridges (Waters, Ireland) for preconcentration and clean-up of sample prior to the LC-MS/MS analysis. Samples were then analyzed on a Thermo TSQ Quantum Ultra EMR (Thermo Fisher Scientific, USA) triple stage quadrupole instrument with a heated electrospray ion source (HESI) operated in positive mode at 3.5 kV ionization voltage. An Agilent eclipse Plus C18, 2.1 × 150 mm, 5 μm column (USA) was used and the gradient program can be found in Appendix A, Table A1.
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