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20 0 0 0 10 A) 15 A) 20 A) 25 B) 50 ml A) B) A) GSTrap FF, 1 ml B) HiTrap Benzamidine FF (high sub), 1 ml Mr 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 4 5 6 7 8 9 Gel: Lane Lane Lane Lane 1. 2. 3. 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.

Lanes 1 and 7. Lane 2. Lane 3. Lane 4. 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.

For greater capacity, use GSTPrep 16/10 FF columns or pack Glutathione Sepharose 4 Fast Flow into a suitable column (see Appendix 3). GSTrap FF columns can be used with a syringe, a peristaltic pump or a chromatography system. Enzyme-specific recognition sites are often included to allow the removal of the GST tag by enzymatic cleavage when required. Thrombin is commonly used for enzymatic cleavage, and must, subsequently, be removed from the recombinant product. HiTrap Benzamidine FF (high sub) 1 ml or 5 ml columns provide simple, ready-made solutions for this process (see page 54).

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