The molecular weight of the T2 channel and the number of bound DDM molecules were calculated using triple-detection SEC (Gimpl et al., 2016 (link)). 60 μL of T2 in 20 mM Tris pH 7,5, 150 mM NaCl, 5 % glycerol, and 0.03 % DDM (before concentration) at 5 mg/mL were injected onto a home-packed SD200 10/300 column pre-equilibrated with the same buffer at a flow rate of 0.5 mL/min. The column was mounted on the 1260 Infinity Bio-inert high-performance liquid chromatography system (Agilent Technologies), and the elution profiles were successively monitored by UV at 280 nm, by refractive index, and by multiple angle light scattering using an in-line refractometer (Optilab rEX, Wyatt Technology), and a mini DAWN™ TREOS system equipped with a quasi-elastic light scattering module (Wyatt Technology). The refractive index increment (dn/dc) of the T2 channel was estimated using the SEDFIT software (http://www.analyticalultracentrifugation.com/default.htm ) and we used a dn/dc of 0.1435 mL/g for DDM. Data were analyzed using the ASTRA 7.0 software.
1260 infinity bio inert
Manufactured by Agilent Technologies
The 1260 Infinity Bio-inert is a liquid chromatography system designed for high-performance liquid chromatography (HPLC) applications. It features a bio-inert flow path, which means the wetted parts are made of materials that are compatible with biological samples and minimize sample-material interactions. The 1260 Infinity Bio-inert is suitable for a wide range of HPLC applications, including analysis of proteins, peptides, and other biomolecules.
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Lab products found in correlation
Glycan mapping, by Agilent Technologies (2 mentions)
1260 fld spectra, by Agilent Technologies (2 mentions)
Chemstation, by Agilent Technologies (2 mentions)
Minidawn treos system, by Wyatt Technology (1 mentions)
Optilab rex, by Wyatt Technology (1 mentions)
Superdex 200 5 150 column, by GE Healthcare (1 mentions)
Superdex 200 5 150 increase column, by Cytiva (1 mentions)
6430 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Resource q column, by GE Healthcare (1 mentions)
Chemstation software, by Agilent Technologies (1 mentions)
9 protocols using 1260 infinity bio inert
1
Molecular Weight and DDM Binding of T2 Channel
2
Analytical SEC for Saposin Nanoparticle Screening
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All small-scale screenings, to monitor the formation of SapNPs and to follow the reconstitution of MPs, were evaluated using analytical SEC. A Superdex 200 (SD200) 5/150 home-packed column (GE Healthcare) equilibrated with detergent-free 1× PBS was used on the 1260 Infinity Bio-inert high-performance liquid chromatography system (Agilent Technologies). The system operates at 10 °C and is equipped with an auto-sampler allowing the consecutive auto-injection of more than 200 samples. In addition, an inline dual detection system permits the parallel detection of the UV absorption at multiple wavelengths and fluorescence (λexcitation = 280 nm and λemission = 350 nm). Typically 50 μl of sample were injected on the gel filtration column and analyzed in duplicates.
In order to estimate the efficiency of a saposin/lipid pair to form SapNPs, we calculated the percentage of the saposin converted to SapNPs using the formula [(1 – (the area under the free saposin peak at a given lipid to saposin molar ratio ÷ the area under the free saposin peak at a lipid to saposin molar ratio of 0)) ∗ 100]. From these calculations, two heat maps were generated for lipid:saposin molar ratios of 6 and 12 using matplotlib (Hunter, 2007 ).
In order to estimate the efficiency of a saposin/lipid pair to form SapNPs, we calculated the percentage of the saposin converted to SapNPs using the formula [(1 – (the area under the free saposin peak at a given lipid to saposin molar ratio ÷ the area under the free saposin peak at a lipid to saposin molar ratio of 0)) ∗ 100]. From these calculations, two heat maps were generated for lipid:saposin molar ratios of 6 and 12 using matplotlib (Hunter, 2007 ).
3
Analytical Gel Filtration of Proteins
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The proteins and protein complexes were analyzed by analytical gel filtration using a Superdex 200 5/150 column (GE Healthcare) and the 1260 Infinity Bio-inert high-performance liquid chromatography system (Agilent Technologies) at 10 °C. The system and column were equilibrated in 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM TCEP and 30 μL of each sample was injected by an auto sampler. The system was run at 0.2 mL/min for 20 min and the elution profile was recorded by a UV detector.
4
SEC-SAXS Characterization of Proteins
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Samples were loaded into a 96-well auto-sampler plate prior to injection into a pre-equilibrated size exclusion-coupled small-angle X-ray scattering (SEC-SAXS) system at the center LiX Beamline of the NSLS-II located at Brookhaven National Laboratory (BNL)(39 (link), 40 (link)). An Agilent 1260 Infinity Bio-Inert high-performance liquid chromatography (HPLC) system with an auto-sampler was used for sample injection and SEC. The beamline was configured to a 1.1 ΰA X-ray wavelength and 1.5mm path-length to obtain the relevant wave-vector, Q = 4sin(θ)/λ, where 2θ is the scattering angle and λ is the X-ray wavelength, yielding a q-range from 0.006 to 3.2 ΰA. A Superdex 200 increase 5/150 column (Cytiva) was equilibrated with buffers and used to separate proteinaceous species using a flow-rate of 0.35 mL/min during 2 s X-ray exposures over the course of 15-minute SAXS data collections. SAXS/WAXS images were radially integrated before being background subtracted using LIX specific pytho packages py4xs and lixtools. Subtracted profiles were exported and imported for analysis in BioXTAS RAW (RAW) (41 (link)).
5
Neonicotinoid Residue Analysis in Beetles
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Topical imidacloprid and thiamethoxam acetone solutions were measured by directly injecting, or diluting and then injecting, 10 µL onto an Agilent Technologies (Santa Clara, California) 1260 infinity bio-inert high-performance liquid chromatograph coupled to a 6430 triple quadrupole mass spectrometer (LC–MS/MS). Dilutions were made using acetonitrile. Instrument details and parameters followed the previous work of Gross et al.57 (link). The limit of detection (LOD) for both AIs was 0.0025 ng/µL.
Acetone rinses followed the same analytical methods as above. Frozen beetles stored at test termination were examined to ensure each beetle used for the neonicotinoid/metabolite residue analyses was intact. Individual beetles with missing limbs were not included. Due to the decreased number of intact beetles, three replicates were used per treatment. Nine beetles were rinsed in 9 mL of acetone for 10 min, i.e., 1 mL per beetle. The 1 mL acetone samples were concentrated to 0.2 mL in acetonitrile, and then 10 µL was injected onto the LC–MS/MS. The neonicotinoid metabolites analyzed included imidacloprid olefin, imidacloprid urea, imidacloprid, 5-hydroxy, and thiamethoxam degradate (CGA-355190)/thiamethoxam urea.
Acetone rinses followed the same analytical methods as above. Frozen beetles stored at test termination were examined to ensure each beetle used for the neonicotinoid/metabolite residue analyses was intact. Individual beetles with missing limbs were not included. Due to the decreased number of intact beetles, three replicates were used per treatment. Nine beetles were rinsed in 9 mL of acetone for 10 min, i.e., 1 mL per beetle. The 1 mL acetone samples were concentrated to 0.2 mL in acetonitrile, and then 10 µL was injected onto the LC–MS/MS. The neonicotinoid metabolites analyzed included imidacloprid olefin, imidacloprid urea, imidacloprid, 5-hydroxy, and thiamethoxam degradate (CGA-355190)/thiamethoxam urea.
6
PfGSK3 Enzymatic Characterization
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An amount of 0.5 mg/ml of PfGSK3 was incubated at 37°C with 2 mM ATP or cAMP and 10 mM MgCl2 overnight. The samples were subsequently dialyzed at 4°C overnight in 50 mM Tris pH 8.0, 20 mM NaCl, 0.5 mM TCEP. The samples were analyzed by analytical ion exchange chromatography using a Resource Q column (GE Healthcare) and the 1260 Infinity Bio-inert high-performance liquid chromatography system (Agilent Technologies) at 10°C. The system and column were equilibrated in 50 mM Tris pH 8.0, 20 mM NaCl, 0.5 mM TCEP. 100 µl of sample was injected and eluted with an increasing concentration of NaCl. The system was run at 0.2 ml/min ad the elution profile was analyzed by UV fluorescence detector with absorbance at 280 nm and emission at 350 nm.
7
HPLC Analysis of Procainamide-Labeled Glycans
8
Quantification of Amino Acid Derivatives
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For experiments in Figure 1 , 8 μl of bead eluate in 50:50 MeOH:borate buffer was combined with 4 μl of freshly prepared derivatization solution comprising 5 mg/ml NBD-Cl (4-chloro-7-nitro-1,2,3-benzoxadiazole) in MeOH and heated to 60° for 70 min on a PCR block. 7-nitro-1,2,3-benzoxiadiazole (NBD) derivatized amines were analyzed on an HPLC system (Agilent Infinity 1260 Bio-Inert) with a reversed-phase C18 column (Agilent Peptide Mapping, 2.1 × 150 mm, 2.7 μm particle size) and fluorescence detector (Agilent 1260 FLD Spectra, 470 nm excitation, 530 nm emission). The instrument was operated using a manual injector, the column was kept at 40°C, and samples were applied to overfill a 5 µl sample loop. Mobile phase A was 95:5 H2O:MeCN containing 10 mm ammonium acetate, and mobile phase B was 95:5 MeCN:H2O containing 5 mm ammonium acetate. For Figure 1 , GABA and glutamate were separated using an optimized gradient as follows: 0–20 min, 10−100% B; 20–25 min, 100% B; 25–30 min, 100−10% B. Peak identities were confirmed by running authentic standards, and areas were determined by integration in Agilent Chemstation software.
9
HPLC Analysis of Procainamide-Labeled Glycans
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An Agilent HPLC system (Infinity 1260 Bio-inert) equipped with a fluorescence detector (Agilent 1260 FLD Spectra, 310 nm excitation, 370 nm emission), amide HILIC column (Agilent Glycan Mapping, 2.1 mm × 150 mm, 2.7 μm particle size) and manual injector was used for analysis of procainamide-labeled glycans. 10 μL glycan digest was injected to overfill a 5 μL home-cut sample loop and the column was kept at 40°C. Mobile phase A was 100 mM ammonium formate, pH 4.4, mobile phase B was 100% acetonitrile. Samples were separated with a 90-min gradient as follows: 0–60 min, 75–62.5% B; 60–62 min, 62.5–15% B; 62–82 min, 15% B; 82–85 min, 15–75% B; 85–90 min, 75% B. The flow rate was 0.25 mL/min except during periods of lower % B (62–85 min), when it was reduced to 0.175 mL/min 30 min was allowed between runs for re-equilibration. Peak areas were determined by automatic integration in Agilent ChemStation software with the following settings: tangent skim mode, new exponential; tail peak skim height ratio, 5.00; front peak ski height ratio, 5.00; skim valley ratio, 20.00; baseline correction, advanced; peak to valley ratio, 500. Peaks eluting between 19 and 60 min were considered for analysis.
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