HisTrap FF crude Kit

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Table 3. Ni Sepharose 6 Fast Flow is compatible with the following compounds, at least at the concentrations given. Reducing agents* 5 mM DTE 5 mM DTT 20 mM ß-mercaptoethanol 5 mM TCEP 10 mM reduced glutathione Denaturing agents 8 M urea † 6 M guanidine hydrochloride † Detergents 2% Triton X-100 (non-ionic) 2% Tween 20 (non-ionic) 2% NP-40 (non-ionic) 2% cholate (anionic) 1% CHAPS (zwitterionic) Additives 500 mM imidazole 20% ethanol 50% glycerol 100 mM Na 2 SO 4 1.5 M NaCl 1 mM EDTA ‡ 60 mM citrate ‡ Buffer substances 50 mM sodium phosphate, pH 7.4 100 mM Tris-HCl, pH 7.4 100 mM Tris-acetate, pH 7.4 100 mM HEPES, pH 7.4 100 mM MOPS, pH 7.4 100 mM sodium acetate, pH 4 † * See notes and blank run, page 6. † Tested for 1 week at 40°C. ‡ The strong chelator EDTA has been used successfully in some cases, at 1 mM. Generally, chelating agents should be used with caution (and only in the sample, not the buffers). Any metal ion stripping may be counteracted by addition of a small excess of MgCl 2 . Note that stripping effects may vary with applied sample volume. p. 20
Appendix B Buffers, Mixing Table for 50 ml Buffer Imidazole concen- Phosphate buffer 8x 2 M Imidazole Deionized tration in buffer stock solution pH 7.4 pH 7.4 water mM ml ml ml 0 6.25 0 to 50 ml 10 6.25 0.25 to 50 ml 20 6.25 0.50 to 50 ml 30 6.25 0.75 to 50 ml 40 6.25 1.00 to 50 ml 50 6.25 1.25 to 50 ml 60 6.25 1.50 to 50 ml 70 6.25 1.75 to 50 ml 80 6.25 2.00 to 50 ml 90 6.25 2.25 to 50 ml 100 6.25 2.50 to 50 ml 150 6.25 3.75 to 50 ml 200 6.25 5.00 to 50 ml 250 6.25 6.25 to 50 ml 300 6.25 7.50 to 50 ml 400 6.25 10.00 to 50 ml 500 6.25 12.50 to 50 ml To obtain the imidazole concentration indicated in the first column, mix Phosphate buffer 8 × stock solution, 2 M imidazole and distilled water according to the table. Check pH and adjust to pH 7.4–7.6 if necessary. These buffers will contain 20 mM phosphate, 500 mM NaCl, and the concentrations of imidazole indicated. For one purification, 24 ml of the binding buffer and 8 ml of each elution buffer are sufficient. p. 21
Page 1 and 2: GE Healthcare Instructions 28-4036-
Page 3 and 4: 1. Introduction HisTrap FF crude Ki
Page 5 and 6: histidines, it is essential to incl
Page 7 and 8: phosphate (1×), 500 mM NaCl, and 2
Page 9 and 10: 2. Wash the column according to Col
Page 11 and 12: Storage For storage, fill the HisTr
Page 13 and 14: No histidine-tagged protein in the
Page 15 and 16: 7. Ordering Information Product No.
Page 17 and 18: Appendix A Description & characteri
Page 19: Buffers The phosphate buffer and th

HisTrap FF crude Kit

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