Purified ABCG2 proteins were obtained from Sino Biological Inc. and were then desalinated using the AKTA protein purification system (Cytiva). Subsequently, proteins were dissolved in buffer solution (200 mM HEPES, 2 mM NaCl and 0.5% DMSO) and were conjugated with sodium acetate solution (pH 4.0) using the Biacore T200 system (Cytiva). Following conjugation with human metal-organic framework, an ethanolamine solution (1.0 M; Shanghai Ruji Biological Technology, Co., Ltd.) was used to block the uncoupled proteins at 28˚C for 6 h. The chip was then added to a buffer solution for 10 h. PCA was dissolved in the buffer solution (10 mmol/l HEPES, 150 mmol/l NaCl and 3 mmol/l EDTA) of the mobile phase. The binding constants between ABCG2 proteins and PCA were determined using a multi-cycle model. The flow rate of the mobile phase, the binding time and the dissociation time were set at 30 µl/sec, 240 and 300 sec, respectively.
Akta protein purification system
Manufactured by Cytiva
The AKTA protein purification system is a versatile and automated liquid chromatography system designed for the efficient purification of proteins. It provides a reliable and reproducible platform for the separation and purification of a wide range of biomolecules, including enzymes, antibodies, and other recombinant proteins. The system's core function is to facilitate the purification process through advanced automated features and high-performance fluidic components.
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2 protocols using akta protein purification system
1
Binding Affinity of ABCG2 and PCA
2
Purification of Galectin-1 Protein
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After the Chemical and Mechanical Lysis process, the lysate was submitted to 3 steps of purification by chromatography in an AKTA Protein Purification System (Cytiva) to obtain a buffered protein solution containing only Galectin-1.
The first step is based on affinity chromatography on agarose-lactose columns (Sigma-Aldrich), previously equilibrated with equilibration buffer (1XPBS, 14mM 2-ME, pH 7.4). After injection of the protein solution, the affinity column "binders" were washed and eluted with elution buffer (1X PBS containing lactose and 2-ME pH 7.4).
The protein peak was collected and 20μM of iodoacetamide (Sigma-Aldrich; I1149) was added to the solution, keeping it under incubation at 4 o C, protected from light, overnight.
After this incubation, the solution was subjected to "size exclusion" chromatography (Sephadex G-25, Cytiva) to remove the free salts of iodoacetamide and lactose. The last chromatographic step was the removal of bacterial endotoxins (LPS). To this end, the preparations were subjected to chromatography using LPS affinity resin (PIERCE High-Capacity Endotoxin Removal Resin column -Thermo Scientific). After all the chromatographic steps, the protein concentration was determined by spectrometry (Abs 280nm) and expressed in milligrams of protein per milliliter (mg/mL) and were submitted to sterilizing filtration (0.22 μm PES membrane).
The first step is based on affinity chromatography on agarose-lactose columns (Sigma-Aldrich), previously equilibrated with equilibration buffer (1XPBS, 14mM 2-ME, pH 7.4). After injection of the protein solution, the affinity column "binders" were washed and eluted with elution buffer (1X PBS containing lactose and 2-ME pH 7.4).
The protein peak was collected and 20μM of iodoacetamide (Sigma-Aldrich; I1149) was added to the solution, keeping it under incubation at 4 o C, protected from light, overnight.
After this incubation, the solution was subjected to "size exclusion" chromatography (Sephadex G-25, Cytiva) to remove the free salts of iodoacetamide and lactose. The last chromatographic step was the removal of bacterial endotoxins (LPS). To this end, the preparations were subjected to chromatography using LPS affinity resin (PIERCE High-Capacity Endotoxin Removal Resin column -Thermo Scientific). After all the chromatographic steps, the protein concentration was determined by spectrometry (Abs 280nm) and expressed in milligrams of protein per milliliter (mg/mL) and were submitted to sterilizing filtration (0.22 μm PES membrane).
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