Materials:

Equipments :

1. Peristaltic pump

2. Vacuum pump

3. A UV/Visible spectrophotometer (if elution is not monitored by an HPLC instrument)

Reagents and chemicals :

1. 1. Sephadex G-100

2. Blue dextran 2000

3. Buffer (Phosphate-buffered saline: 50 mM phosphate buffer, pH ~7.0 – 7.4 + 150 mM NaCl)

Glassware, plasticware, and other materials :

1. 1. Gel filtration chromatography column tube along with the adaptors and tubings

2. A column extension (packing reservoir)

3. A filtration unit

4. Glass rod

5. A laboratory stand

6. 0.22 μm filter

7. Buffer reservoir

8. 1.5 ml microfuge tubes for collecting fractions.

9. 1 ml capacity glass cuvettes

Procedure:

Preparation of the gel

1. 1. Determine the bed volume of the column as follows:

   a.  Fix the column tube in a vertical position on a laboratory stand.

   b.  Insert the bottom adapter to the desired level in the column tube, tighten the adapter, and attach the stop plug.

   c.  Fill the column tube with distilled water up to the point the final gel is to be packed.

   d.  Remove the column cap and collect all the water in a measuring cylinder.

   e.  The volume of the collected water gives the column bed volume, V_t.