| Assay Method Information | |
| | Biochemical Activity Assay |
| Description: | Protein was expressed and purified from E. coli BL21 DE3 Rosetta 2 (EMD Millipore) cells using standard techniques. Cells were grown in 2x yeast extract tryptone medium and expression was initiated via the addition of isopropyl -D-1-thiogalactopyranoside. Expression proceeded overnight at 18 ° C. Cells were harvested by centrifugation and subsequently lysed via sonication. Insoluble fraction was removed by centrifugation. Maltose binding protein (MBP) fusion proteins were purified on a dextrin sepharose column (GE Healthcare) and the MBP tag was removed using tobacco etch virus protease overnight during dialysis. Protein was further purified on a heparin column (GE Healthcare) and eluted using a NaCl gradient. Column fraction were pooled and further purified on a Superdex 75 gel filtration column (GE Healthcare). Protein was quantified using 280 nm absorbance. Protein was then flash frozen in liquid nitrogen and stored at 80 ° C. until use. |
| Affinity data for this assay | |
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