Approximately 10 g cooked protein solution was transferred to a centrifuge tube and centrifuged at 5000× g for 10 min at 4 °C using a centrifuge (LYNX4000, Thermo Fisher Scientific, Pittsburgh, PA, USA), then the water from the centrifuge was weighed. The calculation formula was as follows:
Lynx 4000
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Lynx 4000 is a versatile laboratory instrument designed for a range of analytical applications. It is capable of performing sample preparation, separation, and detection tasks. The Lynx 4000 utilizes advanced technology to provide reliable and accurate results, but no further details about its specific intended use or capabilities are provided.
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Lab products found in correlation
7 protocols using lynx 4000
1
Protein Solution Dehydration Protocol
2
Production and Purification of MaAmyB
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Recombinant E. coli strains were grown as 500 ml cultures in 3 l flasks, with 100 μg·ml−1 ampicillin and 0.05% (final concentration) arabinose (Sigma-Aldrich, Zwijndrecht, the Netherlands) for 6 h at 30 °C (220 rpm) and then for 40 h at 18 °C (220 rpm). Cells were collected by centrifugation at 4,250 g for 20 min at 4 °C (Thermo Lynx 4000). Pellets were resuspended in 50 mM Tris-HCl buffer pH 6.8 containing 10 mM CaCl2. Protease inhibitors (Roche Mini EDTA-free Protease Inhibitor, Sigma-Aldrich, Zwijndrecht, the Netherlands) were added and cells were broken by sonication (15 sec at 10,000 Ω, 30 sec cooling, 7x). Cell debris and intact cells were removed by centrifugation at 15,000 g for 20 min at 4 °C. Resulting cell free extracts were immediately used for purification of MaAmyB.
3
Constructing HIMAR1 Transposon Library in S. meliloti
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A HIMAR1 transposon (19 (link)) library in S. meliloti 1021 was created using triparental mating. Wild-type S. meliloti (recipient), E. coli MT616 (helper), and E. coli Sm10λpir (83 (link)) carrying pSAM_DGm (84 (link)) (donor) were grown to late exponential phase and then diluted to an optical density at 600 nm (OD600) of 2, 1, and 1, respectively. Then, the cultures of the defined optical densities were mixed in equal proportions, transferred onto LBMC, and incubated at 30°C for 6 h. The resulting colonies were then collected in saline (0.85% NaCl) and spread equally onto 50 large petri dishes (150 by 15 mm; VWR) containing LBMC Sm Gent agar (LBMC agar supplemented with Sm and Gent) and incubated at 30°C for 72 h to obtain transposon mutants represented by single colonies. All colonies from all 50 large petri dishes were pooled into saline, concentrated using a Sorvall centrifuge (Thermo Scientific Lynx 4000), and stored in aliquots at −80°C after the addition of glycerol to a final concentration of 20%.
4
Large-Scale Protein Expression in Auto-Induction Medium
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Large scale protein expression was performed in auto-induction medium (1% tryptone, 0.5%, yeast extract, 25 mM Na2HPO4, 25 mM KH2PO4, 25 mM (NH4)2SO4, 2 mM MgSO4, 0.05% glucose, and 0.2% α-lactose) supplemented with 100 µg/mL ampicillin. The inocula were prepared in LB medium as above, 15 mL inocula were used to inoculate 1 L of auto-induction medium, cells were allowed to grow at 37 °C, 180 rpm for 4 h, followed by cultivation for 24 h at 30 °C prior to harvesting the cells (6000× g, 20 min, 4 °C; Sorvall Lynx 4000 centrifuge, Thermo Scientific, Waltham, MA, USA).
5
Extraction and Separation of Meat Protein Components
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The different protein components were extracted and separated as described
previously (Saito et al., 1983 (link)), with
some modifications. All protein extractions were performed on samples cooled
on ice and all solutions used were maintained at approximately 4°C.
Briefly, the minced meat (20 g) was added to 100 mL of low salt buffer (0.05
M KCl, 20 mM Tris-HCl, pH 7.5) and homogenized at 5,054×g for 2 min
using an Ultra-Turrax T25 homogenizer (IKA, Staufen, Germany). After
standing at 4°C for 90 min, the samples were centrifuged at
11,962×g for 20 min, using a refrigerated centrifuge (LYNX4000,
Thermo Fisher Scientific). The supernatant (water-soluble protein) was
stored at 4°C until needed. The precipitates were washed twice with
low salt buffer. Subsequently, the pellets were extracted for 18 h at
4°C after homogenization at 5,054×g for 2 min with 100 mL of
high salt buffer (0.6 M KCl, 20 mM Tris-HCl, pH 7.5), followed by
centrifugation at 11,962×g and 4°C for 20 min. The supernatant
(salt-soluble protein) was stored at 4°C. The precipitates were
washed twice with high salt buffer. The final precipitate (insoluble
protein) was collected and stored at 4°C.
previously (Saito et al., 1983 (link)), with
some modifications. All protein extractions were performed on samples cooled
on ice and all solutions used were maintained at approximately 4°C.
Briefly, the minced meat (20 g) was added to 100 mL of low salt buffer (0.05
M KCl, 20 mM Tris-HCl, pH 7.5) and homogenized at 5,054×g for 2 min
using an Ultra-Turrax T25 homogenizer (IKA, Staufen, Germany). After
standing at 4°C for 90 min, the samples were centrifuged at
11,962×g for 20 min, using a refrigerated centrifuge (LYNX4000,
Thermo Fisher Scientific). The supernatant (water-soluble protein) was
stored at 4°C until needed. The precipitates were washed twice with
low salt buffer. Subsequently, the pellets were extracted for 18 h at
4°C after homogenization at 5,054×g for 2 min with 100 mL of
high salt buffer (0.6 M KCl, 20 mM Tris-HCl, pH 7.5), followed by
centrifugation at 11,962×g and 4°C for 20 min. The supernatant
(salt-soluble protein) was stored at 4°C. The precipitates were
washed twice with high salt buffer. The final precipitate (insoluble
protein) was collected and stored at 4°C.
6
Biotinylated Lambda DNA Preparation
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Biotinylated primer was annealed to Lambda DNA (N3011, NEB). The annealing protocol was: 100 μl (500 ng/μl) Lambda DNA was mixed with 1 μM biotinylated primer, and then the sample was incubated at 65 °C for 5 min, and the temperature was slowly decreased to the room temperature for another 45 min. Then 10× T4 DNA ligase buffer and 5 μl T4 DNA ligase (M0202, NEB) were added into the mix. The mix was incubated at 42 °C overnight. After overnight incubation, Buffer A (30% PEG 8000 and 10 mM MgCl2) was added to dilute the mix. The volume of Buffer A was the half volume of the mix. The new mix was incubated at 4 °C with rotation for 1 day, and then centrifuged at 18,000 g for 5 min by a centrifuge (Lynx 4000, THERMO FISHER). Finally, the pellet including the biotinylated Lambda DNA was dissolved by 100 μl TE150 buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), and 150 mM NaCl).
7
Extraction and Purification of Amandin Protein
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The protein was extracted with the slightly modified method [14] (link). To be specific, the defatted apricot kernel flour was extracted using a magnetic stirrer at room temperature for one hour with deionized water (flour/H2O ratio 1:30 w/v). After centrifugation (LXJ-IIB, Shanghai Anting, China) at 6000 g × for 20 min, the supernatant was collected, and the residue was re-extracted once again under the same condition. The supernatants were pooled and filtered through the 0.45 µm cellulose filter (Shanghai Xinya, China), the filtrate was refrigerated at 4 °C overnight (12–14 h), and an off-white flocculent precipitate was recovered after centrifugation using a high-speed freezing centrifuge (LYNX4000, Thermo Fisher, DE) at 4 °C 10,000 g × for 40 min. The collected precipitate containing mostly amandin was then re-suspended in PBS (0.01 M, pH 7.4) and dialyzed with deionized water (9 h, 4 °C, three changes), frozen, and stored at −20 °C for further use.
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