User:Paul Rothenberg/Notebook/Pauls Notebook/2014/06/17

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Dialysis of Apo Mb

I set the Apo Mb prepared yesterday to run dialysis overnight. We will take a UV-VIS spectrum of it tomorrow.

Note -- it had to be moved from the cold room to the fridge in the lab because of cold room maintenance. Will check on it tomorrow.

Purification of CoMb

Andrew and I are going to purify the CoMb we have through the PD-10 column. We are running it with H2O in the same method we have previously.

After collecting excess porphyrin, we ran 25 mL of 1 M NaOH/NaCl to clear the column, and re-equilibrated with 25 mL of H2O before adding the next aliquot of protein.

Preparation of Mb Citrate Buffer

We are also going to prepare a standard Mb Citrate solution to lyophilize. For a 10 mM Citrate (pH 3) buffer,

  1. 1.177 g Citric Acid Monohydrate (MW 210.14 g/mol, 0.0056 mol)
  2. 1.265 g Sodium Citrate (MW 294.1 g/mol, 0.0043 mol)
  3. 1,000 mL H2O

We have 500 mg of KCl already measured out. We put 5 mg of Mb in, and added 20 mL of buffer.

Buffer Recipes

All are 10 mM buffers

  1. Citrate (pH 3)
    1. 0.0056 mol acid (1.177 g Citric Acid Monohydrate)
    2. 0.0043 mol base (1.265 g Sodium Citrate)
    3. Fill volume to 1,000 mL with H2O
  2. Acetate (pH 5)
    1. 0.0034 mol acid (Acetic Acid)
    2. 0.0065 mol base (Sodium Acetate)
    3. Fill volume to 1,000 mL with H2O
  3. Phosphate (pH 7)
    1. 0.005 mol acid (Sodium Phosphate Monobasic)
    2. 0.0049 mol base (Sodium Phosphate Dibasic)
    3. Fill volume to 1,000 mL with H2O
  4. Tris (pH 9)
    1. 1.211 g Tris
    2. 900 mL H2O
    3. Titrate to pH 9
    4. Fill volume to 1,000 mL with H2O