Module 4 : Chromatographic Methods

Lecture 23 : Protein Molecular Weight Determination Using Gel Filtration Chromatography


Materials:

Equipments :

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

    2. 2. Peristaltic pump  

Reagents and chemicals :

  1. 1. Buffer (Phosphate-buffered saline: 50 mM phosphate buffer, pH ~7.0 – 7.4 + 150 mM NaCl) filtered through 0.22 μm filter (prepared in previous experiment)

    2. 2. Protein standards (Note 1)

    a.  Aprotinin, bovine lung (6.5 kDa)

    b.  Cytochrome c, horse heart (12.4 kDa)

    c.  Carbonic anhydrase, bovine erythrocytes (29 kDa)

    d.  Albumin, bovine serum (66 kDa)

    e.  Alcohol dehydrogenase, yeast (150 kDa)

    f.  β-amylase, sweet potato (200 kDa)

    3. Blue dextran 2000 (1 mg/ml )

Glassware, pasticware, and other materials :

  1. 1. A packed Sephadex G-100 column (packed in previous experiment) (Note 2)

    2. 2. 0.22 μm filter

    3. Buffer reservoir

    4. 1.5 ml microfuge tubes for collecting fractions.

    5. 1 ml capacity quartz cuvettes

Procedure:

  1. 1. Mount the column tube on a stable laboratory stand.

    2. 2. Fill the buffer reservoir with the buffer and place it at a height higher than the pump.

    3. Connect the buffer reservoir to the pump and purge the pump with the buffer.

    4. Keep a low flow rate and allow the buffer to ooze out of the pump outlet tube.

    5. Stop the flow and connect the outlet of the pump to the column tube via an injection valve (injection loop of 500 μl volume).

    6. Remove the bottom stop plug of the column.

    7. Start the pump at a flow rate used for packing the column.

    8. Equilibrate the column with the buffer by allowing two bed volumes to flow though the column.

    9. Determine the void volume using blue dextran 2000 as mention in previous lecture.

    10.  Equilibrate the column again with two bed volumes of buffer.

    11.  Weigh 1 mg of each of the standard proteins and dissolve them together in 1 ml gel filtration buffer. This gives a 6 mg/ml protein solution having equal amount of all the six standard proteins.

    12. Filter the standard protein mixture through 0.22 μm filter.

    13. Bring the injector valve into the load mode.

    14. Inject the protein mixture (≤2% of the bed volume) in the injection loop and change the injection valve into the injection mode. Take out the injection syringe.

    15. Collecting 1 ml fractions until the Vt volume is collected.