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In: Chemistry

Write a protocol for making 250mL of Buffer. Buffer 6XHis Wash Buffer (25 ml) 50 mM...

Write a protocol for making 250mL of Buffer.

Buffer 6XHis Wash Buffer (25 ml)

50 mM Phosphate Buffer pH 7.0

300 mM NaCl

1 mM Imidizole

Adjust pH to 7.0 w/NaOH

Solutions

Expert Solution

Column regeneration should be performed when a different protein is being isolated or when there is a significant loss in the yield of protein. If the Ni-NTA Resin loses its blue color the column needs recharging. 1. Wash the resin with 5 column volumes of a solution 20mM sodium phosphate supplemented with 0.5M NaCl, 50mM EDTA at pH 7.0. 2. Wash with 5 column volumes of distilled water to remove EDTA. NOTE: If the loss in yield is suspected to be due to denatured proteins or lipids a more drastic regeneration protocol should be followed. After step 2: A. Elimination of ionic interactions: Wash in batch for approximately 20 minutes in a solution with 1.5M NaCl, follow with a wash with 10 column volumes of distilled water. B. Elimination of precipitated proteins. Wash in batch for at least 2 hours with a solution 1M NaOH, follow with a wash with 10 column volumes of distilled water. C. Elimination of strong hydrophobic interactions: Resuspend the resin in batch with 30% isopropanol and wash for approximately 20 minutes, follow with a wash with 10 column volumes of distilled water. D. Elimination of lipids: Wash in batch for 2 hours with a solution 0.5% of nonionic detergent in 0.1 M acetic acid. Rinse away the detergent with approximately 10 column volumes of 70% ethanol, follow with a wash with 10 column volumes of distilled water. 3. Add 5 volumes of 0.1M nickel sulfate hexahydrate. 4. Wash with 5 column volumes of distilled water. 5. Add 5 column volumes of the binding buffer. The column is now ready for use.

queen_honey_blossom answered 2 years ago
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