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Star Republic: Guide for Biologists

Purification of his-tagged protein --- TALON

1. Harvest cells and lysis cells in 1X Extraction buffer (50 mM sodium phosphate, 0.3 M sodium chloride, pH 8.0). Clear the supernatant. See Expression of protein in bacterial cells for details.

2. Resuspend the TALON resin. Transfer 2-5 ml to a 15/50 ml tube.

3. Centrifuge at 700 g for 2 minutes to pellet the resin.

4. Remove supernatant. Add 10 bed volumes Extraction/Wash buffer (50 mM sodium phosphate, 0.3 M sodium chloride, pH 7.0).

5. Centrifuge to pellet the resin.

6. Repeat the wash once.

7. Add cleared sample to the resin.

8. Gently agitate at room temperature for 20 minutes on a shaker.

9. Centrifuge to pellet the resin.

10. Remove supernatant.

11. Wash with 10-20 bed volumes 1X Extraction/Wash buffer (50 mM sodium phosphate, 0.3 M sodium chloride, pH 7.0).

12. Gently agitate at room temperature for 10 minutes on a shaker.

13. Centrifuge to pellet the resin.

14. Remove supernatant.

15. Repeat the washing steps 11-14.

16. Resuspend the resin in 1 bed volume 1X Extraction/Wash buffer.

17. Transfer the resin to a gravity-flow column with an end-cap in place.

18. Allow the resin to settle. Remove the end-cap and allow the buffer to drain until it reaches the top of the resin bed.

19. Wash the column with 5 bed volumes of 1X Extraction/Wash buffer.

20. Elute the protein with 5 bed volumes of Elution Buffer (Extraction/Wash buffer + 150 mM Imidazole, pH 7).

21. Collect 0.2-0.5 ml fractions.

22. Determine the protein fraction using a UV spectrometer, Bradford assay, or SDS-PAGE gel analysis.