A modified Matyash extraction method was used to extract neutral lipids (Matyash et al. 2008 (link)). Cells were grown overnight in 2 mL of selection media at 30°C with agitation. The overnight culture was then diluted to a starting cell density of OD660 = 0.25 with SC media and incubated at 30°C with agitation until reaching OD660 = 1 (6–9 h, early-log phase). Cell density was converted to cell count and 1.85 × 108 cells were harvested, centrifuged, washed with ddH2O, and stored at −80°C for at least 24 h before extraction. To extract lipids, 150 µL of 0.5 mm glass beads (DNature) and 400 µL isopropanol were added to the cell pellet that was briefly vortexed and incubated overnight at 4°C while shaking at 1000 rpm (ThermoMix, Eppendorf). To evaporate isopropanol, cell pellets were dried in a speed vac for 30 min, followed by the addition of 700 µL of methyl tertiary-butyl ether (MTBE)/methanol (MeOH) (10:3, v/v). Samples were incubated at 4°C while shaking at 1000 rpm (ThermoMix, Eppendorf). After 1 h, 200 µL of water was added to the extract to aid in phase separation and incubated for a further 20 min before phase separation via centrifugation at 13,000 rpm for 10 min. The lipid-containing upper phase was collected into a new tube, and extracts were dried at room temperature using a nitrogen sample concentrator (Techne) and stored at −80°C.
Thermomix
Manufactured by Eppendorf
Sourced in Germany
The Thermomix is a versatile laboratory equipment designed for precise temperature control and mixing of samples. It features a temperature range from -10°C to 100°C and can mix at speeds up to 3,000 rpm. The Thermomix is suitable for a variety of laboratory applications requiring controlled temperature and mixing.
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Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Polyethylene glycol 8000, by Merck Group (1 mentions)
Phage dna isolation kit, by Norgen Biotek (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Trypsin lys c, by Promega (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Ammonium bicarbonate, by Promega (1 mentions)
2 chloroacetamide caa, by Merck Group (1 mentions)
Trypsin, by Promega (1 mentions)
Dl dithiothreitol, by Merck Group (1 mentions)
Shark chondroitin sulfate, by Merck Group (1 mentions)
Uvmini 1240, by Shimadzu (1 mentions)
Pierce detergent removal spin column, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using thermomix
1
Neutral Lipid Extraction from Yeast Cells
2
Soil Fluoride Quantification Protocol
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Soil samples (10 mg) were incubated for 60 min (95 °C, Thermomix, Eppendorf, Warsaw, Poland) supplemented with 1mL 2 N HClO4. After cooling, sodium citrate (2.5 mL) and TISAB II (2 mL) were added to the 0.5 mL of sample. Soil concentrations of F were determined using a potentiometric ion-selective Orion electrode (Thermo Scientific, Warsaw, Poland) according to the work of Gutowska et al. [47 (link)]. The fluoride content in samples was calculated based on the difference of potentials measured in each sample, the sample weight and the concentration of the added standard.
3
Methylation of Fatty Acids for GC-MS Analysis
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The dried FAs were methylated as previously described (Li et al., 2015b (link)), using methanol-sulphuric acid as a methylating reagent. Briefly, 0.5 mL methanol containing 5% concentrated sulphuric acid was used to resuspend dried lipids by 1 h incubation in a 90°C water bath (Thermomix, Eppendorf, Hamburg, Germany). After cooling to room temperature, 0.5 mL of deionized water was added to terminate the derivatization reaction. FAMEs were subsequently extracted with 0.75 mL of n-pentane, centrifuged (10,000 rpm for 10 min) and the upper phase was then collected and dried with a speed vacuum as mentioned above. The dried pellet was resuspended in 200 μL n-hexane and 1 μL was injected for GC-MS analysis.
4
Phage DNA Extraction Protocol
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DNA from phage lysates was extracted by phenol-chloroform and precipitated by 3 M sodium acetate and 100% ethanol as previously described [21 ] or following the manufacturer’s instructions of the Phage DNA Isolation Kit (Norgen Biotek). Briefly, 300 mL high titer lysate (1010 PFU/mL) was precipitated with 10% polyethylene glycol 8000 (Merck, Germany) and 1 M sodium chloride (Merck) followed by centrifugation for 30 min at 10,000 rpm (Sorvall RC 6 Plus with rotor F21S 8 × 50 y, Thermo ScientificTM, USA). The pellet was resuspended in 2–4 mL SM-buffer and treated with 10-fold reaction buffer (100 mM Tris-HCl (pH 7.5), 25 mM MgCl2, und 1 mM CaCl2, Thermo Fisher Scientific, USA), 0.2 mg/mL RNase A (Thermo Fisher Scientific, USA), and 0.002 U/µL DNase I (Thermo Fisher Scientific, USA) followed by incubation overnight at 37 °C and 300 rpm in a Thermomix (Eppendorf, Germany). DNA was isolated using phenol-chloroform extraction and precipitated by 3 M sodium acetate and 100% ethanol. After incubation for 15 min at −80 °C, DNA was pelleted by centrifugation and washed twice with 70% ethanol. The pellet was air-dried and solved in 50–200 µL TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8, Merck, Germany). The concentration was determined using the Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, USA) following the manufacturer’s instructions.
5
In-Gel Trypsin Digestion for Mass Spectrometry
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Bands from SDS–PAGE gel were cut out with a scalpel and destained in 30% (v/v) ACN (Merck: 1.00029.1000), 25 mM ammonium bicarbonate (Sigma: 09830) and incubated in an ultrasonic bath for 30 min. This step was repeated until all gel pieces appeared clear. Afterwards, the gel pieces were dehydrated using 100% (v/v) ACN for 15 min at room temperature. The acetonitrile was removed, and proteins were reduced and alkylated using 10 mM TCEP (Sigma: C4706) and 25 mM 2‐chloroacetamide (CAA, Sigma: C0267) in 50 mM ammonium bicarbonate for 45 min at room temperature in the dark. Gel pieces were dehydrated afterwards as described above, and supernatant was removed. Trypsin/LysC (Promega: V5111) was resuspended in 100 mM ammonium bicarbonate to a concentration of 0.05 μg/μl, and 20 μl (= 1 μg) was added to each vial containing the dehydrated gel pieces. Acetonitrile was added to a final concentration of 10% (v/v), and protease digest was carried out overnight at 37°C and 300 rpm using a Thermomix (Eppendorf). Peptides were extracted in three steps: 1% (v/v) FA (Merck: 5.33002.0050); 60% (v/v) ACN, 1% (v/v) FA; and as last step 100% (v/v) ACN. Extraction was carried out for 15 min using an ultrasonic bath. After each step, the respective supernatant was removed, combined into a tube and freeze‐dried (ScanVac CoolSafe) overnight.
6
Proteomic Sample Preparation Protocol
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Excised gel bands were treated to remove salts, buffers and detergent using 3 washes of 50 mM NH4HCO3 intercalated with gel shrinkages in 50% acetonitrile (ACN). Disulfide bonds were disrupted with 20 mM DTT at 56°C for 30 min and subsequently alkylated in 55 mM chloroacetamide for 30 min at room temperature. A last wash with 50% ACN and 50 mM NH4HCO3 was performed before addition of 50 ng of trypsin (Promega Sequencing Grade) for an overnight digestion on an Eppendorf Thermomix at 30°C. Peptides were then extracted using 2 washes of 1% formic acid intercalated with gel shrinkages in 50% ACN. The two washes were pooled and evaporated before analysis.
7
Quantification of Sulfated Glycosaminoglycans in Synovial Fluid
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Shark chondroitin sulfate, used as a standard, DL-dithiothreitol (DTT), papain from papaya latex, iodacetic acid and DMMB were obtained from Sigma Aldrich. A calibration curve in a range of 0.25–5 μg chondroitin sulfate in water was obtained (UV mini-1240; Shimadzu, Duisburg, Germany) for calculating the concentrations of sulfated glucosamine glycans (sGAG) in synovial fluid samples of patients according to Farndale et al. [25 (link)]. Briefly, synovial fluid samples were diluted 1:10 with a solution of 1 mM Na2H2EDTA, 2 mM DL-DTT and 300 μg/mL papain in 20 mM sodium phosphate buffer (pH = 6.8) to a final volume of 1 mL. The mixture was incubated for 1 h at 60 °C and 300 rpm in a thermomix (Eppendorf, Hamburg, Germany). The reaction was stopped with 1 mL 20 mM iodacetic acid and diluted with 3 mL of 50 mM TRIS/HCl buffer (pH = 8.0). 500 μL of this incubation mix were added to 2 mL of a dimethylenblue solution and the absorption was measured at λ = 525 nm.
8
Enzymatic Characterization of Recombinant EPO
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Protein pellets recovered from culture supernatant and containing moss-produced rhEPO were dissolved in a 100 mM sodium acetate buffer containing 2% SDS (pH 4.0). After 10 min shaking (1,200 rpm, Thermomix, Eppendorf) at 90°C and additional 10 min centrifugation at 15,000 rpm the supernatant was transferred to a fresh 1.5 ml reaction tube. SDS was removed from the samples using Pierce™ detergent removal spin columns (0.5 ml, Thermo Fisher Scientific) according to the manufacturer’s instructions. Total protein concentration was determined using bicinchoninic acid assay (BCA Protein Assay Kit; Thermo Fisher Scientific) following the manufacturer’s instructions. For each analyzed line, 10 µg of total protein were mixed with one unit of α-L-arabinofuranosidase from either Aspergillus niger or a corresponding recombinant version (E-AFASE or E-ABFCJ, Megazyme, Bray, Ireland) and incubated over night at 40°C. In parallel, enzyme-free samples from each moss line were treated under the same conditions.
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