Recombinant sialylated AIM-A1 fragment, consisting of VWF residues 1238–1493 and C-terminal 10x histidine, referred to herein as sAIM-A1, was produced in BHK cells as described [22 (link)]. Media was collected and loaded onto a HisTrap Excel 5-mL column (GE Healthcare, Chicago, IL). sAIM-A1 was eluted using the buffer containing 150mM NaCl, 20mM phosphate, and 500mM imidazole at pH 7.4. The protein was further purified on a Superdex 200 16/600 preparatory grade (pg) gel filtration column in 1X phosphate-buffered saline (PBS). Positive fractions were pooled and concentrated using a Vivaspin20 concentrator unit (Sartorius, Göttengin, Germany) with a molecular weight cut-off of 10 kDa. The protein was analyzed on a 4–20% SDS-PAGE gel stained with GelCode Blue Stain for verification of purity.
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Recombinant Sialylated AIM-A1 Purification
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Corresponding Organization : Lehigh University
Other organizations : University of Georgia
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Variable analysis
independent variables
- Production of recombinant sialylated AIM-A1 fragment in BHK cells
- Purification of sAIM-A1 using HisTrap Excel column
- Elution of sAIM-A1 using buffer containing 150mM NaCl, 20mM phosphate, and 500mM imidazole at pH 7.4
- Further purification of sAIM-A1 on a Superdex 200 16/600 preparatory grade (pg) gel filtration column in 1X phosphate-buffered saline (PBS)
- Concentration of purified sAIM-A1 using a Vivaspin20 concentrator unit with a molecular weight cut-off of 10 kDa
- Analysis of purified sAIM-A1 on a 4–20% SDS-PAGE gel stained with GelCode Blue Stain
- Molecular weight cut-off of 10 kDa for Vivaspin20 concentrator unit
- PH 7.4 for the elution buffer
- No positive or negative controls were explicitly mentioned in the provided information.
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