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24. Using GSTrap FF with a syringe. A: Prepare buffers and sample. Remove the column’s top cap and twist off the end. B: Equilibrate column, load the sample and begin collecting fractions. C: Wash and elute, continuing to collect fractions. 1. Equilibrate the column with 5 column volumes of binding buffer. 2. Apply the sample. 3. Wash with 5–10 column volumes of binding buffer. 4. Elute with 5–10 column volumes of elution buffer. 5. Wash with 5–10 column volumes of binding buffer. It is important to keep a low flow rate during sample loading and elution as the kinetics of the binding interaction between GST and glutathione are relatively slow.

Lane 6. Mr 97 000 66 45 30 20 14 000 000 000 100 400 Low Molecular Weight Calibration Kit, Amersham Biosciences Crude cell culture supernatant, mouse IgG1, diluted 1:11 Flow through, using a peristaltic pump, diluted 1:10 Eluted mouse IgG1, using a peristaltic pump Flow through, using a syringe, diluted 1:10 Eluted mouse IgG1, using a syringe 1 2 3 4 5 6 7 Fig. 15. SDS-PAGE on PhastSystem using PhastGel 10–15, non-reduced, and silver staining. 31 Performing a separation Column: HiTrap Protein G HP, 1 ml Recommended flow rate: 1 ml/min Binding buffer: Dilute buffer concentrate 10-fold Elution buffer: Dilute buffer concentrate 10-fold Neutralization buffer: Add 60–200 µl of neutralization buffer per ml fraction to the test tubes in which IgG will be collected Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris.

Lane 5. Lane 6. Mr 97 000 66 45 30 20 14 000 000 000 100 400 Low Molecular Weight Calibration Kit, Amersham Biosciences Crude cell culture supernatant, mouse IgG1, diluted 1:11 Flow through, using a peristaltic pump, diluted 1:10 Eluted mouse IgG1, using a peristaltic pump Flow through, using a syringe, diluted 1:10 Eluted mouse IgG1, using a syringe 1 2 3 4 5 6 7 Fig. 15. SDS-PAGE on PhastSystem using PhastGel 10–15, non-reduced, and silver staining. 31 Performing a separation Column: HiTrap Protein G HP, 1 ml Recommended flow rate: 1 ml/min Binding buffer: Dilute buffer concentrate 10-fold Elution buffer: Dilute buffer concentrate 10-fold Neutralization buffer: Add 60–200 µl of neutralization buffer per ml fraction to the test tubes in which IgG will be collected Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris.

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