The indicated concentrations of steroids were prepared separately in DMEM/F12 medium with10% charcoal-stripped FBS, and stripped human serum using ethanol as a carrier solvent. Several extraction methods were tested and were found to have variable abilities to disrupt androgen regulation of reporter activity (data not shown). The method that exhibited androgenic activity most similar to unextracted medium was solid phase extraction using Sep-Pak Cartridges column from Waters (Chromatography division Millipore Corporation, MA, USA). The columns were activated with 4 ml of methanol and subsequently washed with 4 ml of deionized water. Standards made separately in DMEM/F12 with 10% charcoal stripped FBS and stripped human serum were dispensed at a volume of 600 μl in the columns, followed by another wash and elution with 2 ml of 100% methanol. Samples were evaporated at 37°C using a thin stream of nitrogen gas and the dried extract was then re-suspended in 300 μl of DMEM/F12 medium with 10% charcoal stripped FBS.
Sep pak cartridges
Manufactured by Waters Corporation
Sourced in United States
Sep-Pak cartridges are solid-phase extraction (SPE) devices designed for sample preparation and cleanup in analytical workflows. They consist of a sorbent material packed into a disposable plastic cartridge, which can be used to selectively retain, separate, and elute target analytes from complex sample matrices.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein concentration assay kit, by Thermo Fisher Scientific (6 mentions)
Isobaric tmt reagents, by Thermo Fisher Scientific (6 mentions)
Lys c protease, by Fujifilm (6 mentions)
Complete protease inhibitor, by Merck Group (3 mentions)
Mass spectrometry grade trypsin, by Thermo Fisher Scientific (3 mentions)
Pierce c18 tip, by Thermo Fisher Scientific (2 mentions)
High select fe nta phospohpeptide enrichment kit, by Thermo Fisher Scientific (2 mentions)
Complete protease and phosphatase inhibitors, by Merck Group (2 mentions)
Lys c protease, by Thermo Fisher Scientific (2 mentions)
Ms grade trypsin, by Thermo Fisher Scientific (2 mentions)
Acetonitrile, by Merck Group (2 mentions)
Tmt reagent, by Thermo Fisher Scientific (2 mentions)
Ang 1 7 elisa kit, by Cusabio (2 mentions)
Tmt six plex reagents, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Merck Group (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Inova 600 mhz nmr spectrometer, by Agilent Technologies (1 mentions)
241 polarimeter, by PerkinElmer (1 mentions)
Micromass q tof ultima esi tof mass spectrometer, by Waters Corporation (1 mentions)
6130 series esi mass spectrometer, by Phenomenex (1 mentions)
40 protocols using sep pak cartridges
1
Steroid Extraction and Androgenic Activity
2
Isobaric Labeling Proteomics Workflow
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The following reagents were employed: Isobaric TMT reagents (Thermo Fisher Scientific), BCA protein concentration assay kit (Thermo Fisher Scientific), Empore-C18 material for in-house made StageTips (3 M), Sep-Pak cartridges (100 mg, Waters), solvents for Liquid chromatography (LC) (J.T. Baker), mass spectrometry (MS)-grade trypsin (Thermo Fisher Scientific), Lys-C protease (Wako), and cOmplete protease inhibitors (Millipore Sigma). Unless otherwise noted, all other chemicals were purchased from Thermo Fisher Scientific.
3
Purification and Identification of Pb Proteins
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Pb malaysia and Pbm∆109 proteins present in the culture supernatant were acid precipitated adding H2SO4 dropwise to pH 3.5 as described21 (link). Precipitated proteins were extracted by agitation for 2 h in 0.1 M sodium citrate buffer pH 5.5 and analyzed in native protein acrylamide gels. Protein lanes were then excised from the gel and analyzed by mass spectrometry (LTQ Orbitrap Fusion MS coupled to 2-dimension nano-UPLC) at the Proteomics Core facility at the University of California, Riverside. Protein searches were performed against Pb malaysia genome predicted protein database.
For analyses of the cleavage site, cleavage assay mixtures after incubation were peptide purified using Sep-Pak cartridges (Waters) and analyzed by mass spectrometry similarly.
For analyses of the cleavage site, cleavage assay mixtures after incubation were peptide purified using Sep-Pak cartridges (Waters) and analyzed by mass spectrometry similarly.
4
Analytical Methods for Chemical Characterization
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Optical rotations were obtained using a Perkin-Elmer 241 polarimeter. IR spectra were recorded on a Bruker Alpha-P FT-IR spectrometer. UV spectra were acquired on an Amersham Biosciences Ultrospec 5300 Pro Spectrophotometer. CD spectra were measured on a Jasco J-715 spectropolarimeter. HR-ESI mass spectra were recorded on a Waters Micromass Q-ToF Ultima ESI-TOF mass spectrometer at the University of Illinois Urbana-Champaign School of Chemical Sciences Mass Spectrometry Laboratory. LC/MS analysis was performed on an Agilent 1200 Series HPLC system equipped with a diode array detector and a 6130 Series ESI mass spectrometer using an analytical Phenomenex Luna C18 column (5 μm, 4.6 mm × 100 mm). All NMR experiments were carried out on a Varian INOVA 600 MHz NMR spectrometer equipped with an indirect detection probe. HPLC purification was carried out on an Agilent 1100 or 1200 Series HPLC system (Agilent Technologies) equipped with a photo diode array detector. C18 Waters Sep-Pak cartridges were used for column chromatography. Merck precoated silica gel F254 plates and reverse-phase (RP)-18 F254s plates were used for TLC. Spots were detected on TLC under UV light or by heating after spraying with anisaldehyde-sulphuric acid.
5
Biomarker Profiling for Systemic Inflammation
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Creatinine (SpinReact. Girona, Spain), and urea (Wienner Lab. Rosario, Argentina) were analyzed by enzymatic based commercial kits, and sodium concentration was analyzed by an autoanalyzer. Plasma copeptin was measured by ELISA (Peninsula Laboratories. San Carlos, CA, USA), previous reverse phase extraction (SepPak cartridges, Waters. Milford, MA). Plasma uric acid was measured by an enzymatic based kit (Sekisui Diagnostics. Kent, UK). Markers of systemic inflammation included IL-1, IL-6, and TGF-beta and were evaluated by ELISA (Abcam. Cambridge, MA, USA).
6
Automated TMT Proteomics Workflow
7
Proteomics and Phosphoproteomics Materials
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Materials used in proteomics and phosphoproteomics assessments: Isobaric TMT reagents (Thermo Fisher Scientific), BCA protein concentration assay kit (Thermo Fisher Scientific), Empore-C18 material for in-house made StageTips (3 M), Sep-Pak cartridges (Waters), Mass spectrometry (MS)-grade trypsin (Thermo Fisher Scientific), Lys-C protease (Wako), High-Select Fe-NTA phospohpeptide enrichment kit (Thermo Fisher Scientific), and cOmplete protease and phosphatase inhibitors (Millipore Sigma). Unless otherwise noted, all solvents used for liquid chromatography (LC) were purchased from J.T. Baker all other chemicals were purchased from Thermo Fisher Scientific.
8
Mass Spectrometry-Based Proteomic Workflow
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Reagents for tissue culture, including DMEM, fetal bovine serum (FBS), penicillin/streptomycin, and phosphate-buffered saline (PBS) were obtained from Gibco. Mass spectrometry-grade trypsin and Lys-C protease were purchased from ThermoFisher Scientific and Wako, respectively. Isobaric TMT reagents and the BCA protein concentration assay kit were from ThermoFisher Scientific. Empore-C18 material for in-house StageTips was acquired from 3 M and Sep-Pak cartridges were purchased from Waters. Sera-Mag Speed Beads (cat. nos. 45152105050350 and 65152105050350) were from GE Life Sciences (Marlborough, MA). All solvents used for liquid chromatography were purchased from VWR. Unless otherwise noted, all other chemicals were purchased from ThermoFisher Scientific.
9
Quantitative Analysis of Omija Beverages
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To prepare the sample, 1 g of freeze-dried pulp, seed, leaf, and stem, and 200 mg of flowers were ground and extracted under sonication with 200 mL of methanol for 2 h. The extracts were centrifuged at 3000 g at 4 °C for 10 min, and the supernatant was filtered with a 0.45 μm PTFE membrane filter (Whatman, New York, NY, USA) for HPLC analysis.
Subsequently, 100 μL of filtrate obtained from each Omija beverage was diluted with 200 μL of MeOH. Thereafter, 200 μL of the diluted sample was loaded on Sep-Pak Cartridges (C18, Waters Corporation, Milford, MA, USA) and then eluted with 3 mL of MeOH. The eluent was analyzed by HPLC.
Subsequently, 100 μL of filtrate obtained from each Omija beverage was diluted with 200 μL of MeOH. Thereafter, 200 μL of the diluted sample was loaded on Sep-Pak Cartridges (C18, Waters Corporation, Milford, MA, USA) and then eluted with 3 mL of MeOH. The eluent was analyzed by HPLC.
10
Isobaric Labeling and MS-based Proteomics
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