Akta avant

Manufactured by GE Healthcare

Sourced in United Kingdom, United States, Canada, Sweden

The AKTA Avant is a versatile liquid chromatography system designed for efficient protein purification. It enables the automated separation and purification of biomolecules, providing precise control over flow rates, gradients, and other parameters. The AKTA Avant is a reliable and flexible tool for researchers and scientists working in the field of protein purification and analysis.

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Lab products found in correlation

27 protocols using akta avant

Digestibility of Peptides 6 and 8

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To determine the digestibility of peptides 6 and 8 in the human digestive tract, the 10 mL samples from the TIM-1 compartments withdrawn at different sampling times, including the initial meal, were lyophilized, resolubilized in 2 mL of sodium phosphate buffer (0.05 M) (pH 7.2), and filtered over 0.45 uM. To detect the peptides in these samples, a HiTrap SP FF cation exchange column (1 mL, GE, Mississauga, ON, Canada) was used on a FPLC system (Akta Avant, GE Healthcare, Baie-D’Urfé, QC, Canada), and 500 uL injections were performed for each sample. The equilibration buffer consisted of sodium phosphate buffer (0.05 M) (pH 7.2). Before each injection, the column was equilibrated with six column volumes of equilibration buffer.
For each run, after the sample injection, the column was washed with 5 column volumes of equilibration buffer, and a linear NaCl gradient was then used until a NaCl concentration of 0.5 M was achieved over 6 column volumes. A second linear gradient was then applied to achieve a 0.9 M NaCl concentration over two column volumes. The column was then washed with equilibration buffer containing 1 M NaCl for 5 column volumes, and finally, it was equilibrated with 6 column volumes of equilibration buffer. The absorbance was recorded at 214 nm.

Cashman-Kadri S., Lagüe P., Fliss I, & Beaulieu L. (2023). Assessing the Activity under Different Physico-Chemical Conditions, Digestibility, and Innocuity of a GAPDH-Related Fish Antimicrobial Peptide and Analogs Thereof. Antibiotics, 12(9), 1410.

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Purification of Ubiquitin and ERD10 Proteins

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Lyophilized ubiquitin (UBQ) was obtained from Sigma Chemical (St. Louis, MO, USA), whereas plant late embryogenesis abundant protein early response to dehydration 10 (ERD10, UniProt P42759) was produced via recombinant expression in Escherichia coli BL21(DE3) Star expression strain and purified as described previously [22 (link)]. In short, purification was carried out through three chromatographic steps: an ion exchange on HiTrap Q FF at pH 9.5 with gradient elution, followed by two gel-filtration steps on Superdex 200 and Superdex 75 columns, on an AKTA Avant (GE Healthcare, Little Chalfont, UK) FPLC system. The purity of the proteins was checked by SDS-PAGE and was found to be at least 98%.

Tompa K., Bokor M, & Tompa P. (2018). The Melting Diagram of Protein Solutions and Its Thermodynamic Interpretation. International Journal of Molecular Sciences, 19(11), 3571.

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SEC Purification of 21t15-TGFRs Complex

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Example 21

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.8 mL/min. A capillary loop was used to inject 2004, of 1 mg/mL of 21t15-TGFRs complex onto the column. The injection was then chased with 1.25 column volumes of PBS. The SEC chromatograph was shown in FIG. 31. There were two protein peaks, likely representing a monomer and dimer forms of 21t15-TGFRs.

US11518792B2. Multi-chain chimeric polypeptides and uses thereof (2022-12-06). HCW Biologies, Inc. [US]. Inventors: Hing Wong [US].

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Bispecific Antibody Production in Expi293 Cells

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BsAb was generated by transient co-transfection of four expression plasmids encoding anti-mBCMA heavy/light chain and anti-mCD3 heavy/light chain into Expi293 cells (Thermo Fisher Scientific). Heavy chains (HC) and light chains (LC) were transfected with a DNA ratio (by weight) of 1:1:1:1 for HC_BCMA:HC_CD3:LC_BCMA:LC_CD3. Six days after transfection supernatant was harvested and purified on an AKTA Avant (GE Healthcare). The supernatant was loaded on MabSelect Sure LX (GE Healthcare) column, washed with Phosphate Buffered Saline (PBS), eluted in 100 mM pH 3.6 sodium citrate buffer, neutralized, and dialyzed into PBS. The bispecific quality was examined by intact LC-MS after PNGase F (Prozyme or New Englab Biolabs) deglycosylation. Control KLH/CD3 bsAb was generated following the same protocol as described above.

Meermeier E.W., Welsh S.J., Sharik M.E., Du M.T., Garbitt V.M., Riggs D.L., Shi C.X., Stein C.K., Bergsagel M., Chau B., Wheeler M.L., Bezman N., Wang F., Strop P., Bergsagel P.L, & Chesi M. (2021). Tumor Burden Limits Bispecific Antibody Efficacy through T-cell Exhaustion Averted by Concurrent Cytotoxic Therapy. Blood Cancer Discovery, 2(4), 354-369.

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Recombinant Equine Nrf2, Keap1, and Rev Proteins

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cDNA encoding full-length equine Nrf2 (Genbank No. XM_008536608.1) and Keap1 (Genbank No. XM_023645423.1) were PCR-amplified from equine macrophage cells and subcloned into a pET vector containing C-terminal His6, and a PEGX6P vector with C-terminal GST tag separately. cDNA encoding EIAV rev was subcloned into a modified PET29a with a C-terminal MBP. For protein expression, BL21 (DE3) competent or Rosetta (DE3) competent Escherichia coli cells were used. Protein expression in E. coli cells was induced with 0.3 mM isopropyl-D-thiogalactopyranoside (IPTG) overnight at 18°C. His-tagged Nrf2 protein and MBP-tagged Rev protein were purified with nickel affinity (HisTrap; GE Healthcare), eluted with imidazole, and desalted into binding buffer. GST-tagged Keap1 was purified on glutathione-Sepharose 4B (GE Healthcare), eluted with 10 mM reduced glutathione in 50 mM Tris, pH 8.0, and 150 mM NaCl. Protein desalinations were further performed using Akta Avant (GE Healthcare). Purity of the proteins was monitored at all stages of the purification process, and proteins were visualized using Coomassie blue staining.

Wang Y., Ma G., Wang X.F., Na L., Guo X., Zhang J., Liu C., Du C., Qi T., Lin Y, & Wang X. (2022). Keap1 recognizes EIAV early accessory protein Rev to promote antiviral defense. PLoS Pathogens, 18(2), e1009986.

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Determining Molecular Weight Distribution

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Molecular weight distribution was performed on the hydrolysate sample by gel permeation chromatography on a Superdex™ Peptide 10/300 GL column (GE Healthcare, Baie-D’Urfé, Qc, Canada) using a FPLC system (Akta Avant, GE Healthcare, Baie-D’Urfé, Qc, Canada). The column was calibrated according to the manufacturer’s guidelines with proteins of known molecular weight (reference samples). The mobile phase consisted of 50 mM sodium phosphate buffer containing 150 mM of NaCl at pH 7.0. A mixture of glycine (1.00 mg/mL), aprotinin (0.200 mg/mL), ribonuclease A (0.200 mg/mL), and bovine serum albumin (0.650 mg/mL) (Sigma, Oakville, ON, Canada) was injected (0.500 mL) on the column for the calibration. This yielded a near linear correlation between the retention time and the log of the molecular mass of proteins in the range of 6512 Da to 75 Da. Briefly, the hydrolysate was solubilized at 1 mg/mL with the mobile phase and then injected (0.500 mL) on the column. Samples were eluted (isocratic) at a flow rate of 0.8 mL/min. Proteins/peptides were detected by monitoring the absorbance at 214 nm.

Offret C., Fliss I., Bazinet L., Marette A, & Beaulieu L. (2019). Identification of A Novel Antibacterial Peptide from Atlantic Mackerel belonging to the GAPDH-Related Antimicrobial Family and Its In Vitro Digestibility. Marine Drugs, 17(7), 413.

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Protein A Chromatographic Purification

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Lab scale chromatographic separations were performed using an AKTA Avant chromatographic system (GE Healthcare) and a 1.0 cm inner diameter (I.D.) Omnifit Benchmark chromatography columns (Omnifit Ltd) packed with MabSelect SuRe Protein A resin (GE Healthcare) to a 20 cm bed height. After equilibration with two column volumes (CVs) of 20 mM sodium phosphate pH 7.2 the column was loaded to 15 g binding antibody/L with clarified mammalian cell culture fluid. Binding antibody concentration was determined by summation of the bispecific and FcFc homodimer titers. Columns were washed and protein eluted via a pH 6.0–3.0 gradient in 40 mM acetate, 500 mM calcium chloride. Bispecific and FcFc titers were measured using a POROS A 20 μm column (2.1 mm × 30 mm, 0.1 mL) cat#2-1001-00, and Fc*Fc* titers were measured by loading the flowthrough over a POROS G 20 μm column (2.1 mm × 30 mm, 0.1 mL) cat#2-1002-00.

Smith E.J., Olson K., Haber L.J., Varghese B., Duramad P., Tustian A.D., Oyejide A., Kirshner J.R., Canova L., Menon J., Principio J., MacDonald D., Kantrowitz J., Papadopoulos N., Stahl N., Yancopoulos G.D., Thurston G, & Davis S. (2015). A novel, native-format bispecific antibody triggering T-cell killing of B-cells is robustly active in mouse tumor models and cynomolgus monkeys. Scientific Reports, 5, 17943.

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Purification of 21t15-TGFRs complex

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Example 24

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.8 mL/min. A capillary loop was used to inject 2004, of 1 mg/mL of 21t15-TGFRs complex onto the column. The injection was then chased with 1.25 column volumes of PBS. The SEC chromatograph was shown in FIG. 45. There were two protein peaks, likely representing a monomer and dimer forms of 21t15-TGFRs.

US11672826B2. Methods of treating aging-related disorders (2023-06-13). HCW Biologies, Inc. [US]. Inventors: Hing Wong [US].

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Gel Filtration Analysis of 21t15-TGFRs Complex

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Example 45

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.8 mL/min. A capillary loop was used to inject 2004, of 1 mg/mL of 21t15-TGFRs complex onto the column. The injection was then chased with 1.25 column volumes of PBS. The SEC chromatograph was shown in FIG. 53. There were two protein peaks, likely representing a monomer and dimer forms of 21t15-TGFRs.

US11730762B2. Methods for stimulating proliferation or differentiation of an immune cell with a multi-chain chimeric polypeptide (2023-08-22). HCW Biologies, Inc. [US]. Inventors: Hing Wong [US].

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Gel Filtration Purification of 7t15-21s Complex

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Example 63

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A capillary loop was used to inject 2004, of 1 mg/mL of 7t15-21s complex onto the column. The injection was chased with 1.25 column volumes of PBS.

US11730762B2. Methods for stimulating proliferation or differentiation of an immune cell with a multi-chain chimeric polypeptide (2023-08-22). HCW Biologies, Inc. [US]. Inventors: Hing Wong [US].

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