SEC experiments were run on an Akta Pure 25 system (Cytiva). Absorbances were measured at both 280 nm (blue traces) or 254 nm (red traces). Molecular weight markers (Bio-Rad) were used as a reference (grey dotted traces). Samples from SEC experiments were collected and run on SDS-PAGE gels stained with Coomassie brilliant blue.
Akta pure 25 system
Manufactured by Cytiva
Sourced in United States
The AKTA Pure 25 system is a versatile liquid chromatography instrument designed for protein purification and other biomolecular separations. It features a flow rate range of 0.001 to 25 mL/min and a pressure limit of 50 MPa, enabling a wide variety of applications. The system is equipped with advanced detection capabilities, including UV, conductivity, and pH monitoring, to provide comprehensive data on the purification process.
Automatically generated - may contain errors
Lab products found in correlation
Molecular weight marker, by Bio-Rad (1 mentions)
Unicorn software, by Cytiva (1 mentions)
Quantared enhanced chemifluorescent hrp substrate kit, by Thermo Fisher Scientific (1 mentions)
Histrap hp column, by Cytiva (1 mentions)
Infinite f500 plate reader, by Tecan (1 mentions)
Black 96 well plate, by Thermo Fisher Scientific (1 mentions)
Perfect block, by MoBiTec (1 mentions)
5 protocols using akta pure 25 system
1
Size-Exclusion Chromatography Analysis
2
Purifying AAV Vectors via Affinity Chromatography
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HiScale 26/40 column hardware (Cytiva) was packed with POROS™ CaptureSelect™ AAV affinity resin (Thermo Fisher Scientific) to a bed height of 7.5 cm for a final column volume of 40 mL. The column was loaded with 400 mL of equilibration buffer at pH 7.5 for each experiment containing a combination of iodixanol and AAV-B vector as per Table 1 . Two different AAV-B preparations were used for this study as a demonstration. The column was then eluted via low pH buffer and the eluate fraction was collected into a vessel containing buffer for adjustment to a neutral pH. All steps were performed at a flow rate of 150 cm/hr. Each experiment used distinct but identical POROS CaptureSelect AAV affinity columns from the same lot. An AKTA Pure 25 system with UNICORN software (Cytiva) was used for each purification run.
The pH-adjusted AAV affinity-based purification eluate fractions for experiments containing AAV vector were concentrated and buffer exchanged into formulation buffer via ultrafiltration and diafiltration (UFDF) using hollow fiber filters. The levels of AAVs were estimated via genome titer quantification using digital droplet polymerase chain reaction (ddPCR), and residual iodixanol levels were determined by the LC-MS method described previously.
The pH-adjusted AAV affinity-based purification eluate fractions for experiments containing AAV vector were concentrated and buffer exchanged into formulation buffer via ultrafiltration and diafiltration (UFDF) using hollow fiber filters. The levels of AAVs were estimated via genome titer quantification using digital droplet polymerase chain reaction (ddPCR), and residual iodixanol levels were determined by the LC-MS method described previously.
3
Purification and Characterization of scFv Protein
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HeLa cells (1 × 106 cells in 10-cm dish) were transfected with the pcDNA/ss-scFv vector using PEI. After incubation in 0.5% FBS-DMEM for 24 h, the secreted His6-tagged scFv protein in the culture medium was purified using a HisTrap HP column (Cytiva, Marlborough, MA, USA) with the AKTA pure 25 system (Cytiva).
ELISAs were carried out in 96-well black plates (Thermo Fisher Scientific) with all steps performed at room temperature with the exception of coating that was conducted by incubation with recombinant human CD25-Fc (R&D systems, Minneapolis, MN, USA) overnight at 4 °C. Blocking was performed with 2% Perfect Block (MoBiTec) in PBS (PBS-PB) for 2 h, followed by washing three times with PBS-T and incubation with His6-tagged scFv in PBS-PB for 1 h. The wells were then washed again three times with PBS-T and incubated with a 1000-fold diluted HRP-conjugated α-His tag antibody (Abcam, Cambridge, MA, USA) in PBS-PB for 1 h. Before detection using a QuantaRed Enhanced Chemifluorescent HRP Substrate kit (Thermo Fisher Scientific), the wells were washed three times with PBS-T and then another three times with PBS. The resulting fluorescence signals were measured using an Infinite F500 plate reader (Tecan, Mannedorf, Switzerland) with specific filters (Ex/Em = 535 nm/590 nm).
ELISAs were carried out in 96-well black plates (Thermo Fisher Scientific) with all steps performed at room temperature with the exception of coating that was conducted by incubation with recombinant human CD25-Fc (R&D systems, Minneapolis, MN, USA) overnight at 4 °C. Blocking was performed with 2% Perfect Block (MoBiTec) in PBS (PBS-PB) for 2 h, followed by washing three times with PBS-T and incubation with His6-tagged scFv in PBS-PB for 1 h. The wells were then washed again three times with PBS-T and incubated with a 1000-fold diluted HRP-conjugated α-His tag antibody (Abcam, Cambridge, MA, USA) in PBS-PB for 1 h. Before detection using a QuantaRed Enhanced Chemifluorescent HRP Substrate kit (Thermo Fisher Scientific), the wells were washed three times with PBS-T and then another three times with PBS. The resulting fluorescence signals were measured using an Infinite F500 plate reader (Tecan, Mannedorf, Switzerland) with specific filters (Ex/Em = 535 nm/590 nm).
4
Purification of AAV2-GFP Capsids
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AEX chromatography column, buffers, and method were similar to those described in the literature.3 ,5 (link),6 (link),7 (link) All of the experiments were performed using an AKTA Pure 25 system (Cytiva) connected to a fraction collector. The affinity-purified AAV2-GFP samples were prepared and loaded onto the AEX column at a capsid load ranging from 4.6E+13 to 4.6E+14 cp/mL. The column was then chased with 5 CV of equilibration buffer. The column would then be washed with a linear gradient of equilibration buffer and salt elution buffer for full to empty (F/E) AAV capsid separation. The column eluate was collected in 1 mL column fractions using a 96 deep well plate and pooled based on the UV A260/A280 ratios of the chromatogram, with the eluted empty capsids having an absorbance peak with A260/A280 < 1 and the full-genome–containing capsids having an absorbance peak with A260/A280 > 1. All of the chromatography steps were performed at 5 CV/min in the downflow direction at ambient temperature. The conductivity values and elution times taken of the peaks from each AEX experiment are the respective values at the A280 peak maxima value.
5
Scalable AAV2-GFP Purification Protocol
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AAV2-GFP was produced using suspension-adapted HEK293 cells in a 3 L shake flask with 1 L working volume for each GFP genome size (1.9, 3.4, and 4.9 kb) for 3 separate transfection conditions. The frozen cell culture harvest was thawed, 0.45-μm bottle top filtered using a vacuum, and then loaded onto a 5 mL AAVX Pre-Packed column at a flow rate of 5 mL/min (1-min residence time). All of the affinity capture chromatography experiments used POROS GoPure AAVX Pre-Packed 5-mL columns (Thermo Fisher Scientific, Bedford, MA). The affinity capture chromatography experiments were performed using an AKTA Pure 25 system (Cytiva, Marlborough, MA) connected to a fraction collector. The affinity capture chromatography step would first be a 5 column volume (CV) wash with equilibration buffer, followed by loading, a 5 CV wash with equilibration buffer, and then elution with 5 CV of elution buffer similar to those described in the literature.7 (link) The eluate would then be titrated to pH 8 and sampled.
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