ECD-Fc proteins were produced in Expi293F cells using transfection conditions described above. Following harvesting of cell conditioned media, 1 M Tris, pH 8.0 was added to a final concentration of 20 mM. Ni-NTA Agarose (QIAGEN) was added to ∼5% conditioned media volume. 1 M sterile PBS, pH 7.2 (GIBCO) was added to ∼3X conditioned media volume. The mixture was stirred overnight at 4°C. Ni-NTA Agarose beads were collected in a Buchner funnel and washed with ∼300 mL protein wash buffer (30 mM HEPES, pH 7.2, 150 mM NaCl, 20 mM imidazole). Beads were transferred to an Econo-Pak Chromatography column (Bio-Rad) and protein was eluted in 15 mL of elution buffer (30 mM HEPES, pH 7.2, 150 mM NaCl, 200 mM imidazole). Proteins were concentrated using Amicon Ultracel 10K filters (Millipore) to a volume of ∼0.5 mL and absorbance at 280 nm was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) to determine protein concentration.
Amicon ultracel 10k filters
Manufactured by Merck Group
Sourced in United States
Amicon Ultracel 10K filters are laboratory equipment used for filtration. They are designed to separate molecules or particles based on their size, with a molecular weight cut-off of 10,000 Daltons.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (8 mentions)
Econo pak chromatography column, by Bio-Rad (3 mentions)
Ni nta agarose, by Qiagen (3 mentions)
Expifectamine, by Thermo Fisher Scientific (3 mentions)
Expi293 expression medium, by Thermo Fisher Scientific (3 mentions)
Sterile pbs, by Thermo Fisher Scientific (1 mentions)
Econo pac chromatography column, by Bio-Rad (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
5 protocols using amicon ultracel 10k filters
1
Purification of ECD-Fc Proteins from Expi293F Cells
2
Recombinant Ly6a-Fc Protein Production
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Ly6a-Fc was produced in Expi293F suspension cells grown in Expi293 Expression Medium (Thermo Fisher Scientific) in a 37 °C, 5% CO2 incubator with 130 rpm shaking. Transfection was performed with Expifectamine according to the manufacturer’s instructions (Thermo Fisher Scientific). Following harvesting of cell-conditioned media, 1 M Tris, pH 8.0, was added to a final concentration of 20 mM. Ni-NTA Agarose (QIAGEN) was added to ∼5% conditioned media volume. 1 M sterile PBS, pH 7.2 (GIBCO), was added to ∼3X conditioned media volume. The mixture was stirred overnight at 4 °C. Ni-NTA Agarose beads were collected in a Buchner funnel and washed with ∼300 mL protein wash buffer (30 mM HEPES, pH 7.2, 150 mM NaCl, 20 mM imidazole). Beads were transferred to an Econo-Pak Chromatography column (Bio-Rad) and protein was eluted in 15 mL of elution buffer (30 mM HEPES, pH 7.2, 150 mM NaCl, and 200 mM imidazole). Proteins were concentrated using Amicon Ultracel 10 K filters (Millipore) and absorbance at 280 nm was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) to determine protein concentration.
3
Synthesis of Neomycin-Biotin Conjugate
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Neomycin trisulfate (contains 6 amino groups per 1 molecule) was taken in a quantity of 0.6 mg (0.66 µM), and biotinamidohexanoyl-6-aminohexanoic acid N-hydroxysuccinimide ester was taken in a quantity of 3 mg (5 µM). Then both preparations were added to 50 µL of C2H5OH and incubated for 1 h. The activated biotin derivative was added in excess to minimize the number of unreacted neomycin amino groups. Next, 200 µL of the 10% BSA solution (0.3 µM) was added to the reaction mixture and incubated for 30 min. One BSA molecule contains more than 30 surface amino groups [23 (link)]. That is, 0.3 µM BSA is able to bind more than 9 µM of activated biotin. The resulting conjugate was purified using Amicon Ultracel 10 K filters (Millipore, USA), centrifuging the reaction mixture at 10,000× g for 15 min. The solution passed through the filter containing the target neomycin–biotin conjugate, whereas BSA, which has bound excess biotin, remained on the filter. The same methodology was used to synthesize streptomycin-biotin conjugate.
4
Production and Purification of LY6A-Fc Protein
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LY6A-Fc was produced in Expi293F suspension cells grown in Expi293 expression medium (Thermo Fisher Scientific) in a 37°C and 5% CO2 incubator with shaking at 130 rpm. Transfection was performed with Expifectamine according to the manufacturer’s instructions (Thermo Fisher Scientific). Following harvesting of cell-conditioned media, 1 M tris (pH 8.0) was added to a final concentration of 20 mM. Ni–nitrilotriacetic acid (NTA) agarose (QIAGEN) was added to ∼5% conditioned media volume. One-molar sterile PBS (pH 7.2; GIBCO) was added to ∼3× conditioned media volume. The mixture was stirred overnight at 4°C. Ni-NTA agarose beads were collected in a Buchner funnel and washed with ∼300 ml of protein wash buffer [30 mM Hepes (pH 7.2), 150 mM NaCl, and 20 mM imidazole]. Beads were transferred to an Econo-Pac chromatography column (Bio-Rad), and protein was eluted in 15 ml of elution buffer [30 mM Hepes (pH 7.2), 150 mM NaCl, and 200 mM imidazole]. Proteins were concentrated using Amicon Ultracel 10K filters (Millipore), and absorbance at 280 nm was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) to determine protein concentration.
5
Recombinant Ly6a-Fc Protein Production
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Ly6a-Fc was produced in Expi293F suspension cells grown in Expi293 Expression Medium (Thermo Fisher Scientific) in a 37 °C, 5% CO2 incubator with 130 rpm shaking. Transfection was performed with Expifectamine according to manufacturer’s instructions (Thermo Fisher Scientific). Following harvesting of cell conditioned media, 1 M Tris, pH 8.0 was added to a final concentration of 20 mM. Ni-NTA Agarose (QIAGEN) was added to ~5% conditioned media volume. 1 M sterile PBS, pH 7.2 (GIBCO) was added to ~3X conditioned media volume. The mixture was stirred overnight at 4 °C. Ni-NTA Agarose beads were collected in a Buchner funnel and washed with ~300 mL protein wash buffer (30 mM HEPES, pH 7.2, 150 mM NaCl, 20 mM imidazole). Beads were transferred to an Econo-Pak Chromatography column (Bio-Rad) and protein was eluted in 15 mL of elution buffer (30 mM HEPES, pH 7.2, 150 mM NaCl, 200 mM imidazole). Proteins were concentrated using Amicon Ultracel 10K filters (Millipore) and absorbance at 280 nm was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) to determine protein concentration.
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