Materials:

Equipments :

1. 1.  HPLC instrument with binary pump and spectrophotometric detection

2.  Vacuum pump

3.  Centrifuge

Reagents and chemicals :

1. 1.  Acetonitrile (HPLC grade)

2.  Methanol (HPLC grade)

3.  Deionized water

4.  Trifluoroacetic acid (TFA)

Glassware, plasticware, and other items :

1. 1.  Two 1 liter bottles

2.  Two 1 liter measuring cylinders

3.  Filter assembly

4.  47 mm PTFE membrane filter (0.45 μm )

5.  Pipettes

6.  Pipette tips

7.  Reversed-phase C18 column

8.  Guard column

9.  13 mm syringe filter (0.22 μm )

10.  100 μl  injection syringe

11.  1.5 ml  microfuge tubes

12.  Spanners of appropriates sizes (for fixing the column and the guard column)

Reagent preparation

Solvent A (0.1% TFA in deionized water)

1. 1.  Measure 1 litre of deionized water using a measuring cylinder.

2.  Add ~400 – 500 ml of this in the bottle labeled ‘Solvent A'.

3.  Add 1 ml  TFA to the bottle and shake the bottle slowly to achieve mixing.

4.  Add rest of the water into the bottle and cover the bottle.

5.  Filter the solvent through 47 mm PTFE membrane filter (0.45 μm).

Solvent B (0.1% TFA in acetonitrile)

1. 1.  Measure 1 litre of HPLC grade acetonitrile using a measuring cylinder.

2.  Add ~400 – 500 ml of this in the bottle labeled ‘Solvent B'.

3.  Add 1 ml TFA to the bottle and shake the bottle slowly to achieve mixing.

4.  Add rest of the acetonitrile into the bottle and cover the bottle.

5.  Filter the solvent through 47 mm PTFE membrane filter (0.45 μm).

Protein/peptide solution

1. 1.  Weigh 1 mg of the given protein or peptide and transfer it into a 1.5 ml microfuge tube.

2.  Add 1 ml deionized water to dissolve the protein/peptide.

3.  If dissolution is not complete, add 25 μl acetonitrile and vortex the sample.

4. Filter the sample through a 0.22 μm filter to remove any particular material or insoluble protein/peptide. Alternatively, centrifuge the sample at 16,000 × g for 5 minutes and collect the supernatant.