An ultra-high-performance liquid chromatography system (UltiMate 3000 BioRS, Thermo Scientific) coupled to an FLD-3400RS fluorescence detector (Thermo Scientific) was used for performing the analytical-scale SE-UHPLC fractionation. Flucelvax monovalent drug substance (3mg) was applied to a Superose 6 Increase 10/300 GL column, (Cytiva, Cat# 29-0915-96) equilibrated in running buffer (0.37 g/L monobasic potassium phosphate, 1.29 g/L disodium phosphate, 0.1 g/L magnesium chloride, 0.2 g/L potassium chloride and 8.0 g/L sodium chloride, pH 7.2) and run through the column at a rate of 0.12 mL/min (column temperature 30°C). Antigen peaks were collected as 16 x 1mL fractions with an AKTA pure chromatography system (Cytiva) and pooled to represent HA species: high molecular weight (MW) HA oligomers A (fractions 1-5), high MW HA oligomers B (fractions 9 and 10), HA trimer (fraction 12) and HA monomer (fraction 15). In-line fluorescence data (FLD excitation 280nm, emission 345nm) was assessed using Chromeleon 7.2 software package. The molecular weight of SE-UHPLC separated HA species was determined using gel filtration standards (Bio-RAD, cat# 151-1901). The 4 fractions including Monovalent control antigens were normalized by total protein nitrogen before being assayed via SRID and ELISA methods.
Akta pure chromatography system
Manufactured by Cytiva
Sourced in United States, New Zealand
The AKTA pure chromatography system is a versatile and automated liquid chromatography system designed for a wide range of purification applications. It is capable of performing various chromatographic techniques such as ion exchange, size exclusion, and affinity chromatography. The system is equipped with advanced monitoring and control features to ensure precise and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
F9 c 96 well plate fraction collector, by Cytiva (2 mentions)
Synergy mx, by Agilent Technologies (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Superose 6 increase 10 300 gl column, by Cytiva (1 mentions)
Ultimate 3000 biors, by Thermo Fisher Scientific (1 mentions)
Cd 1 mice, by Charles River Laboratories (1 mentions)
Superdex 200 increase column, by Cytiva (1 mentions)
5 protocols using akta pure chromatography system
1
Analytical-scale SE-UHPLC Fractionation of Influenza Vaccine Antigens
2
SARS-CoV-2 Spike Ectodomain Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The coding sequence of the SARS-CoV-2 spike ectodomain amino acids 1–1215 (NCBI Accession # MN908947), was modified (Pallesen et al., 2017 (link)), and placed in frame with a Thrombin cleavage site, T4 foldon trimerization motif (Tao et al., 1997 (link)), a Strep tag II (Schmidt and Skerra, 2007 (link)) and a FLAG tag (Hopp et al., 1988 (link)). The sequence was codon optimized for human cell expression, and cloned into pcDNA 3.1. Recombinanat SP was expressed in HEK 293 Expi cells and purified on an AKTA Pure chromatography system (Cytiva, Marlborough, MA, USA). Recombinant NP was expressed and purified using a similar regimen. (Supplementary Materials and Methods ).
3
Murine Plasma Analysis of Cy5-siRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Murine plasma was collected 45 min after a 1 mg/kg intravenous injection of Cy5-labeled siRNA conjugate into 6–8 week old, male CD-1 mice from Charles River (Wilmington, MA). Plasma was filtered (0.22 µm) and then injected into an AKTA Pure Chromatography System from Cytiva (Marlborough, MA) with three inline Superdex 200 Increase columns (10/300 GL). Fractionation was done at 0.3 ml/min using Tris running buffer (10 mM Tris-HCl, 0.15 M NaCl, 0.2% NaN3) into 1.5 ml fractions with a F9-C 96-well plate fraction collector (Cytiva). Cy5 fluorescence was measured in fractions (100 µl) in black, clear-bottom, 96-well plates from Greiner-Bio-One (Kremsmunster, Austria, REF 675096) on a SynergyMx from Biotek (Winooski, VT) at a gain of 120, excitation 642/9.0, emission 675/9.0. Fraction albumin-bound conjugate was determined by taking the sum of fluorescence intensity for fractions associated with albumin elution divided by the sum of fluorescence intensity for all fractions collected. Albumin-associated fractions were determined by running known protein standards through the SEC system and examining A280 of eluate from each of the fractions.
4
Recombinant TNfxB-His and NfxB-His Protein Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Open-read fragments of tnfxB or nfxB were cloned into pMAL-c5x with a 6 × His tag. The recombinant plasmid was then introduced into E. coli BL21(DE3) pLysS and cultured in 500 mL Luria-Bertani broth at 30°C with shaking, until the OD600 reached 0.4–0.5. Then, 0.25 mM isopropyl β-D-1-thiogalactopyranoside was added to induce the TNfxB-His or NfxB-His protein, and the cells were cultured for 20 h at 16°C. The bacterial cells were harvested by centrifugation at 5,000 × g for 20 min and the pellets were resuspended in 50 mM phosphate buffer (pH 7). The harvested cell suspension was split by a low-temperature ultra-high pressure cell disrupter (JN-2.5C, Juneng Biol) and was further centrifuged at 4°C, 13,000 × g, for 20 min. The supernatant was filtered through a 0.45-µm filter and the His-tagged protein was enriched and purified using an AKTA pure chromatography system (Cytiva) with a HisTrap HP purification column. Finally, desalting columns were used to remove the salts from proteins, which were stored at 4°C. Protein concentrations were determined using a bicinchoninic acid assay (Thermo Fisher Scientific). Protein purity was verified using SDS-PAGE on a 12.5% polyacrylamide gel.
5
Plasma Cy5-siRNA Conjugate Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Murine plasma was collected approximately 45 min after a 1 mg/kg intravenous injection of Cy5-labeled siRNA conjugate. Plasma was filtered (0.22 μm) and then injected into an AKTA Pure Chromatography System (Cytiva) with three inline Superdex 200 Increase columns (10/300 GL). Fractionation was done at 0.3 mL/min using Tris running buffer (10mM Tris-HCl, 0.15M NaCl, 0.2% NaN3) into 1.5 mL fractions with a F9-C 96-well plate fraction collector (Cytiva). Cy5 fluorescence was measured in fractions (100 μL) in black, clear-bottom, 96-well plates (Greiner-Bio-one REF 675096) on a SynergyMx (Biotek) at a gain of 120, excitation 642/9.0, emission 675/9.0. Fraction albumin-bound conjugate was determined by taking the sum of fluorescence intensity for fractions associated with albumin elution divided by the sum of fluorescence intensity for all fractions collected. Albumin-associated fractions were determined by running known protein standards through the SEC system and examining A280 of eluate from each of the fractions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!