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

From OpenWetWare
Jump to navigationJump to search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png


Hb buffer exchange


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