Protocol:Agar gel electrophoresis

Agar is a gelatinous substance derived from the cell wall of red marine algae. It is composed of a matrix of cross-linked molecules with spaces between them. In an electric field the haemoglobin molecules will move through the matrix so that the migration rates depend on the size and shape of the molecules as well as the charge. This means that smaller, linear molecules with high electrical charge will move through the gel at a faster rate.

Agar gel electrophoresis is not a satisfactory screening technique for haemoglobinopathies, because it cannot distinguish many abnormal haemoglobins from Hb A. However, it can separate the C group into three fractions: HbC, O-Arab, and Hb E plus HbA2. The method can also distinguish Hb S from Hb D, Hb F from Hb A, Hbs Little Rock, Rainier and Bethesda from Hb A, and Hb H from Hb I.

Reagents

 * 1) Stock buffer &mdash; Citrate buffer, pH 5.9:
 * Dissolve 73.5 g tri-sodium citrate (Na3C6H5O7.2H2O) in approximately 700 mL distilled water. Adjust the pH to 5.9 using 0.5 M citric acid (10.5 g per 100 mL). Approximately 34 mL will be required. Make the solution up to 1 litre with distilled water, and store at 4 °C.
 * 1) Working buffer:
 * Dilute stock buffer 1 in 5 with distilled water. The pH will be 6.
 * 1) Gel:
 * Pre-prepared made Titan IV Citrate Agar plates, provided by Helena Laboratories U.K.
 * 1) Bromophenol blue &mdash; for staining of the gel:
 * Dissolve 20 mg bromophenol blue in 200 mL distilled water containing 2 mL glacial acetic buffer.

Equipment

 * 1) Power supply capable of delivering 30 – 40 mA is necessary (Vokan SAE 2761, Vokan 400)
 * 2) A Southern horizontal electrophoresis tank.
 * 3) Gel slot, a device for making sample application wells.

Method

 * 1) Using the gel slot, 5 slots are made on the gel, mid-way between anode and cathode.
 * 2) Using a capillary tube enough haemolysate is delivered to fit each slot on the gel.
 * 3) 700 ml of the working buffer is poured into the electrophoresis tank.
 * 4) Wicks made from Whatman No. 3 chromatographic paper, are placed along the bridges of the electrophoresis tank. The gel is placed along the wicks and is held in position using extra wicks made of Whatman No.3 chromatographic paper in order to ensure contact with the buffer.
 * 5) A current 30 mA approximately (40 V) is applied across the gel.
 * 6) Electrophoresis is run at 4 °C.
 * 7) After approximately 3 hours, separation is achieved.
 * 8) The agar plate is stained for 20 minutes with the bromophenol blue stain and then rinsed with distilled water.