The full-length NTR of M. mazei strain Gö1 (MM2353) was cloned into pET28a, resulting in a cleavable N-terminal 6xHis-tagged fusion protein. Protein was produced in BL21pRIL Escherichia coli cells upon induction with 0.2 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) at 22 °C. Cells were disrupted by sonication in 20 mM Tris-HCl buffer, pH 7.6, containing 300 mM NaCl, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride. The crude extract was clarified by centrifugation at 40,000× g for 1 h. The enzyme was purified using nickel-nitriloacetic acid (Ni-NTA) chromatography (GE Healthcare) and digested with thrombin (EMD Chemicals, San Diego, CA, USA) in buffer, 20 mM Tris-HCl pH 8, 150 mM NaCl, and 2 mM CaCl2, at room temperature overnight. The last step of purification involved gel filtration chromatography (Sephacryl S-300) using 10 mM Tris-HCl pH 7.6, 100 mM NaCl, and 2 mM 2-mercaptoethanol buffer. For protein molecular weight estimation by size exclusion chromatography, the column was calibrated with the Gel Filtration Standard mixture from Bio-Rad.
Gel filtration standard mixture
Manufactured by Bio-Rad
The Gel filtration standard mixture is a set of proteins with known molecular weights that can be used to calibrate and validate gel filtration chromatography systems. The mixture is designed to provide a range of molecular weights for testing the performance and resolution of gel filtration columns.
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5 protocols using gel filtration standard mixture
1
Purification of Recombinant M. mazei NTR Protein
2
Determining Recombinant Myo-IDH Mass
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The native molecular mass of recombinant, 6xHis-tagged myo-IDH was determined using size-exclusion chromatography Superdex 200 Increase 10/300 GL column connected to BioLogic DuoFlow system (BioRad, Hercules, USA). The column was calibrated with BioRad gel filtration standard mixture (cat # 1511901), and the running buffer was 10 mM sodium pyrophosphate used for the enzymatic assay. Fractions of 0.5 ml were collected for monitoring the myo-IDH activity in the elution profile.
For MALS analysis, samples of enzymatically active myo-IDH (0,5–2 mg/ml) were injected onto an Agilent Biosec-3 column (4.6x300 mm) at a flow rate of 0.3 ml/min in 50 mM phosphate buffer (pH 7.0) and 300 mM NaCl at 15°C. The column was coupled with static light scattering (miniDAWN, Wyatt Technology), differential refractive index (Shodex RI-501) and Agilent 1260 Infinity II UV (Agilent Technologies) detectors. Data were analyzed using ASTRA software (Wyatt Technology).
For MALS analysis, samples of enzymatically active myo-IDH (0,5–2 mg/ml) were injected onto an Agilent Biosec-3 column (4.6x300 mm) at a flow rate of 0.3 ml/min in 50 mM phosphate buffer (pH 7.0) and 300 mM NaCl at 15°C. The column was coupled with static light scattering (miniDAWN, Wyatt Technology), differential refractive index (Shodex RI-501) and Agilent 1260 Infinity II UV (Agilent Technologies) detectors. Data were analyzed using ASTRA software (Wyatt Technology).
3
Thermal Stability of BCD085 Antibody
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Example 12
Determination of the Aggregation Stability of BCD085 Under Thermal Stress
BCD085 preparation of 9 mg/ml in PBS was heated for 6 hours at a temperature of 50° C. Aggregation after the thermal stress was evaluated by size-exclusion HPLC. Chromatographic analysis was performed on Agilent 1100 system with Tosoh TSK-Gel G3000SWXL column (7.8 mm×30 cm, Cat. No. 08541) and Tosoh TSKgel Guard SWXL pre-column (6.0 mm×4.0 cm, 7 μm particles, Cat. No. 08543). Isocratic elution with mobile phase containing 50 mM sodium phosphate buffer and 0.3 M NaCl (pH 7.0) was performed under 0.5 ml/min flow rate with the detection at 214 nm and 280 nm wave lengths. Antibody samples were diluted with PBS (pH 7.5) to a concentration of ˜1 mg/ml. Injection volume was 10 μl. Gel filtration standard mixture (Bio-Rad, Cat. No. 151-1901) was used to calibrate the column prior to the test.
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4
Size Distribution Analysis of Aβ Peptides
5
SARS-CoV-2 S1 Protein and IFN-γ Effects
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Calu-3 and A549-hACE2 cells were singly treated or co-treated with spike S1 (100 ng/ml) and IFN-γ (500 U/ml) for 24 h in serum-depleted medium. Cytosolic extract from 80 to 90% confluent monolayers (3 mg protein) was applied to a Superose-6 FPLC column and 0.5 ml fractions eluted at a flow rate of 0.25 ml/min in buffer containing 50 mM Tris-HCl (pH 7.6), 50 mM NaCl, and 1 mM DTT. Gel filtration standard mixture (Bio-Rad, #1511901) was used as molecular weight standards. 40–45 μL of fractions was used in immunoblots; 6-9 μL was used in RNA EMSA.
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