Affinity Chromatography by Amersham Biosciences

By Amersham Biosciences

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4. Lane Lane Lane Lane 5. 6. 7. 8. Lane 9. ExcelGel SDS Gradient 8–18%, Coomassie Blue staining Low Molecular Weight Calibration Kit (LMW) Clarified E. coli homogenate expressing SH2-GST fusion protein Flow-through from GSTrap FF (Fraction 2) SH2 GST-tag cleaved, washed off with binding buffer through both columns (Fraction 6) as above (Fraction 7) as above (Fraction 8) Elution of thrombin, HiTrap Benzamidine FF (high sub) Elution of GST-tag and some non-cleaved SH2-GST, GSTrap FF (Fraction 21) as above (Fraction 22) Fig.

The purity of the eluted IgY may be improved by using gradient elution with, for example, a linear gradient 0–100% elution buffer over 10 column volumes, followed by elution with 100% elution buffer for a few column volumes. To increase capacity, connect several HiTrap IgY Purification HP columns in series. A HiTrap column can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system such as ÄKTAprime. Reuse of HiTrap IgY Purification HP depends on the nature of the sample.

GSTrap FF 1 ml 10–12 mg recombinant GST/column 4 ml/min Prepacked column, ready to use. GSTrap FF 5 ml 50–60 mg recombinant GST/column 15 ml/min Prepacked column, ready to use. GSTPrep FF 16/10 >200 mg recombinant GST/column >10 ml/min Prepacked column, ready to use. Glutathione Sepharose 4 Fast Flow 10–12 mg recombinant GST/ml medium 450 cm/h* For packing high performance columns for use with purification systems and scaling up. Glutathione Sepharose 4B 8 mg horse liver GST/ml medium 75 cm/h* For packing small columns and other formats.

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