User:NYOUSHA Yousefi/Notebook/Protein Engineering/2013/02/05

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Title

Hb buffer exchange

Objective

The wild type Hb's buffer was exchanged to a new buffer containing 1g manitol, 0.5g PEG, and 50ml phosphate pH 7

Procedure 1

  1. Column chromatogrophy was rinsed with 25ml of distilled water and 25ml of the new buffer solution containing 0.5g PEG, 1g manitol,and 50ml of phosphate buffer, pH 8
  2. 2.5ml of WT Hb and 2.5ml of the new buffer were added to the column and this 5ml was collected in a new tube (2% manitol, 1% PEG, WT Hb, pH 7 phosphate)

Objective 2

Extracting bacteria cells

Procedure 2

  1. The bacteria expressing Mn and Ni were allowed to grow in media for 6 hours after addition of IPTG, which is a signal that stimulates protein expression
  2. The media was divided and balanced into four smaller flasks and they were placed in centrifuge at 4500rpm, 4 degree C for 20 min
  3. After 20 min, the supernatant of the samples was discarded and the pellets including WT Ni and Mn Hb, BL-21(DE3) were resuspended in Tris pH 9 buffer separatley
  4. These samples were stored at -80 degree C freezer