Crude protein extracts of P. insidiosum, including soluble antigen from broken hyphae (SABH; containing intracellular proteins) and culture filtrate antigen (CFA; containing secreted proteins), were prepared according to the methods described by Chareonsirisuthigul et al [41 (link)]. Briefly, 100 ml Sabouraud dextrose broth was inoculated from an actively growing P. insidiosum colony and incubated, with shaking (~150 rpm), at 37°C for 10 days. The organism was killed with 0.02% Thimerosol (Sigma). Hyphae were collected by filtration on a 0.22-μm-pore-size membrane (Durapore, Merck Millipore), and ground in a mortar with pre-cooled distilled water (1.5 g hyphae per 30 ml water). Supernatant, following centrifugation (10,000 x g) of the cell lysate at 4°C for 30 min, was filtered through a 0.22-μm-pore-size membrane (Durapore, Merck Millipore). Both filtered supernatant (SABH) and cell-free broth (CFA) were 100-fold concentrated by ultrafiltration (10,000 molecular weight cut-off membrane; Amicon Ultra 15M, Merck Millipore). Protein concentration was measured by Bradford’s assay [42 (link)]. SABH and CFA were stored at -20°C until use.
Durapore
Manufactured by Merck Group
Sourced in United States, Germany, Ireland, Japan, United Kingdom, Slovenia
Durapore is a high-performance membrane filter product from Merck Group. It is designed for a range of laboratory filtration applications. Durapore membranes are made from polyvinylidene fluoride (PVDF) and feature a porous structure.
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57 protocols using durapore
1
Preparation of Crude Protein Extracts from P. insidiosum
2
Isolation of Sponge-Associated Microbes
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The sponges P. ficiformis (sample ID: 1Biotec2_S07) and S. foetidus (sample ID: 1Biotec2_S06) were collected on 25 May 2013, by SCUBA diving in Milos, Greece (N36.76759° E24.51422°), at 5–7 m depth. Sponge tissues (5 ml each) were washed with sterile-filtered seawater, passed through a 100 μm Nitex cloth (Hartenstein, Germany) and transported to the laboratory in glycerol solution (15% v/v) at -20°C until further processing. A total of 10 L seawater (sample ID: Biotec_SW) was collected from the vicinity of the sponges. Within 2–3 h after collection, seawater was filtered consecutively through 100 μm Nitex (Hartenstein), 5 μm durapore (Merck-Millipore), and finally through 0.22 μm durapore membrane filters, which were then frozen at -20°C.
Sponge samples of A. aerophoba were collected in the Mediterranean Sea from a depth of 5 m (Piran, Slovenia), on 07 May 2013. Upon transport back to the laboratory, samples of pinacoderm and mesohyl were separated using a sterile scalpel blade. One scalpel blade was used per each sample to prevent cross-contamination between samples. Microbial cells were enriched from the different sponge tissues by differential centrifugation (Fieseler et al., 2006 (link)). Microbes from P. ficiformis and S. foetidus samples were prepared using the same protocol. Fractions of sponge-associated prokaryotes (SAPs) were frozen at -80°C in 15% glycerin.
Sponge samples of A. aerophoba were collected in the Mediterranean Sea from a depth of 5 m (Piran, Slovenia), on 07 May 2013. Upon transport back to the laboratory, samples of pinacoderm and mesohyl were separated using a sterile scalpel blade. One scalpel blade was used per each sample to prevent cross-contamination between samples. Microbial cells were enriched from the different sponge tissues by differential centrifugation (Fieseler et al., 2006 (link)). Microbes from P. ficiformis and S. foetidus samples were prepared using the same protocol. Fractions of sponge-associated prokaryotes (SAPs) were frozen at -80°C in 15% glycerin.
3
HPLC mobile phase optimization
4
Quetiapine Fumarate In Vitro Release
5
Axenic Growth of Oceanicaulis alexandrii
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Oceanicaulis alexandrii was the last contaminant of N. adriaticus NF5 and N. piranensis D3C enrichment cultures prior to their isolation into pure culture, and has previously been shown to be able to grow in co‐culture with all three investigated Nitrosopumilus strains (Bayer et al., 2019b). In this study, the alphaproteobacterium was grown axenically in SCM medium amended with 100 μM amino acids (alanine, aspartic acid, glutamic acid, serine) in the dark at 30°C without shaking, and growth was monitored by flow cytometry (Marie et al.,1999 ). After reaching late exponential growth, O. alexandrii was starved for 3 days and then transferred into nine 20 ml flasks containing fresh SCM medium (1% inoculum). To three replicates, Nitrosopumilus‐derived SPE‐DOM and SPE‐DOM from culture medium controls was added, respectively, and three replicates served as no substrate control incubations. Methanol extracts (1.2 ml each) were dried down in a SpeedVac (Eppendorf, Concentrator Plus), subsequently re‐dissolved in SCM medium and sterile‐filtered through 0.1 μm syringe filters (Millipore Durapore, 25 mm). DOM extracts were added to 20 ml of O. alexandrii culture, resulting in a 20X concentration as compared to the initial DOC concentrations retained on SPE columns (approx. 40 μM of Nitrosopumilus‐derived DOM and 20 μM of medium blank SPE‐DOM).
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Oceanicaulis alexandrii was the last contaminant of N. adriaticus NF5 and N. piranensis D3C enrichment cultures prior to their isolation into pure culture, and has previously been shown to be able to grow in co‐culture with all three investigated Nitrosopumilus strains (Bayer et al., 2019b). In this study, the alphaproteobacterium was grown axenically in SCM medium amended with 100 μM amino acids (alanine, aspartic acid, glutamic acid, serine) in the dark at 30°C without shaking, and growth was monitored by flow cytometry (Marie et al.,
6
Particle Solution Filtration and Dialysis
7
Endo-metaproteomics for Microbial Communities
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For analyses of endo-metaproteomes, biomass was collected onto 0.22-µm pore-size hydrophilic PVDF, Durapore® (Millipore) filters. For the coastal endo-metaproteome, 4 L of the microbial inoculum was collected prior to the start of the experiments. For the endo-metaproteome of the jelly-OM degrading microbial community, 500 mL was collected from each jellyfish treatment (J1, J2 and J3) at the peak of prokaryotic abundance (at 32 h) in Experiment 1 and 1 L at the decay phase (at 84 h) in Experiment 2. For the endo-metaproteome of the control treatments, 2 L was collected from each flask (C1, C2 and C3) at the peak of the bacterial abundance (at 32 h) and 3 L at the decay phase (at 84 h). Protein extraction from collected cells was performed as described in the Additional File 1 .
8
Effective Helminth Ova Extraction
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Three batches of five reclaimed water samples (500 mL) each were seeded with 1, 5, or 10 A. lumbricoides eggs (five replicates per sample). Reclaimed water not seeded was used as negative control in each batch. To retain the helminth ova, seeded reclaimed water was filtered through a 47 mm diameter Durapore® filter with a light mesh of 0.65 μm (Millipore Corp., Bedford, MA, USA). This filter was mechanically and enzymatically digested by adding 1 g of glass beads (425–600 μm diameter), 920 μL of NET 10 (10 mM NaCl, 10 mM EDTA, and 10 mM Tris HCl), 40 μL of proteinase K (30 mg mL−1), and 40 μL of 10% sodium dodecyl sulfate (SDS) (Sigma-Aldrich, Poole, Dorset, UK). Then, samples were subjected to three cycles of freezing with liquid nitrogen for 5 s followed by thawing in a 56°C water bath for 5 min and vortexed at maximum speed in a Vortex Genie 2 machine for 2 min (MoBio Laboratories, Inc., Solana Beach, CA, USA). The samples were centrifuged at 8,000 rpm for 1 min and the supernatant (containing DNA) was transferred to new tubes. DNA was extracted by conventional phenol : chloroform : isoamyl alcohol purification (25 : 24 : 1) and ethanol precipitation and further eluted in 50 μL of bdW.
9
Quantitative Metabolite Analysis and 13C Tracing
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For metabolite measurements and 13C labeling experiments, an amount proportional to 1 mL*OD600 of the culture broth was transferred onto a 0.45 μm pore size Durapore filter (Millipore) and vacuum-filtered. For metabolite measurements, the filter with cells was immediately transferred after filtration into 4 mL of 20°C acetonitrile/methanol/water (2:2:1) to quench metabolism and 200 μL of a uniformly 13C labeled E. coli metabolite extract were added as internal standard45 (link). 13C labeling experiments were performed immediately after vacuum-filtration on the filter, as described previously46 (link). Specifically, cells on the filter were first washed with fresh, preheated (37°C) acetate M9 medium for 10 sec and 13C labeling was initiated by changing the washing solution to preheated (37°C) M9 medium containing uniformly 13C labeled acetate. After each labeling step, the filter was transferred into 4 mL of 20°C acetonitrile/methanol/water (2:2:1) for quenching. To extract metabolic intermediates, the filter was kept in this solution at -20°C for 1 h. Then, the cell debris was removed from the extracts by centrifugation (4°C, 10,000 rpm, 10 min), the supernatants transferred into new tubes and dried to complete dryness.
10
Quantitative Metabolite Analysis and 13C Tracing
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