Protocol:Starch gel electrophoresis

Although considered outdated by many, the classical starch gel electrophoresis method for separation of Hb Fractions at alkaline pH is still valuable under many circumstances. Because of the low price is applicable in laboratories with limited resources. Moreover, home made preparation and good separation, detection of low expression fractions down to 0.5% and approximate visual estimation make this method still attractive when more sophisticated HPLC systems produce artifacts such as high HbF due to overlapping with glycated fractions.

This method of Starch Gel Electrophoresis pH 8.6 is adapted from the original Smithies method.

Reagents

 * 1) Acetic Acid,
 * 2) Boric acid,
 * 3) Sodium chloride
 * 4) TRIS
 * 5) Hydrogen peroxide 30% (H2O2).
 * 3,3’-Dimethoxybenzidine (o-Diansidine) (C14H16N2O2).
 * 1) Ethylene Diamine Tetra Acetate (Na2EDTA).
 * 2) Sodium Acetate Trihydrate ( CH3COONa-3H2O)
 * 3) Methanol, 96%, technical.
 * 4) Saponin, white pure.
 * 5) Starch (potato), hydrolysed Smithies (Stärke nach Smithies, Carl Roth GmbH),
 * 6) (C6H10O5) Brunschwig.

Solutions

 * 1) Smitthies-buffer (stock)
 * For 2 Liter: 219 g Tris, 11.68 g Na2EDTA, 70 g boric acid, pH = 8.6.
 * Adjust pH with boric acid.
 * 1) Smitthies Gel buffer:
 * Dilute the Smitthies-buffer 1:20; by adding 12.5 ml of Smitthies-buffer to 250 ml distilled water.
 * 1) Smitthies running buffer:
 * Dilute the Smitthies-buffer 1:7; by adding 50 ml of Smitthies-buffer to 300 ml distilled water.
 * 1) Saponin Lysis buffer
 * For 0,5 liter: 2,2 gram TRIS, 0.5 g Boric Acid, 0,136 g Na2EDTA, 1.5 g Saponin, 0.5 g KCN. Adjust to pH 8.6 with Tris or boric acid.
 * 1) Sodium Acetate, 1 M
 * Add 272 g. of Na-acetate (CH3COONa-3H2O) in 1700 distilled water. Adjust to pH 4.7 with 80-90 ml Acetic Acid.
 * 1) Phosphate Buffered Saline
 * Add 7 g NaCl to 1 L of 0.1 M phosphate buffer pH 7,4.
 * 1) Staining solution
 * Add under fume cupboard a ‘tea spoon’ of dimethoxybenzidine to 150 ml 96% technical methanol, 50 ml sodium-acetate and 2 ml H2O2. Mix for 15 minutes. This staining solution can only be used once.

Preparing a starch-gel
Home made glass plates are needed, provided with thin glued edges 3mm high of the same material. Plates of 16 x 15 cm are suitable for an average of 10-15 samples per run.


 * 1) Purchase a cooling plate to accommodate one or more glass plates (see two examples on Fig. 1).
 * 2) Place the empty tray on the cooling system kept at 5oC.
 * 3) Weigh 22.7 g of Starch and transfer it to a flame resistant round bottom flask.
 * 4) Prepare and add the Smitthies gel buffer to the starch.
 * 5) Mix well before heating on a flame, and keep heating while shaking vigorously until bubbles appear.
 * 6) When the boiling point is almost reached connect the flask to a vacuum system and keep the flask for 30 seconds shaking under vacuum until air bubbles are removed.
 * 7) Pour the gel carefully of the glass tray from the center to the corners.
 * 8) Let the gel solidify for 30 – 40 minutes before loading the samples.

Preparation of the samples

 * 1) Mix the blood sample kept in the refrigerator by inverting the tube a few times
 * 2) Pipet 200 ul of blood into a tube filled with PBS.
 * 3) Centrifuge for 5 minutes at 3000 rpm.
 * 4) Remove the PBS by vacuum suspiration.
 * 5) Add 100 ul of saponin lysis buffer to the pellet.

Vortex the tubes several time, until the lysis of the erythrocytes is complete.

Loading and running of the starch-gel

 * 1) Cut, with a scalpel and a ruler, a straight incision across the width of the gel, 3.5 cm from the edge.
 * 2) Cut for every sample a loading paper of convenient proportions (3MM Whatman).
 * 3) Take a loading paper with a pair of tweezers and immerge it in the tube containing the lysate.
 * 4) Remove the excess of lysate by placing the soaked paper on a sheet of filter paper for a few seconds.
 * 5) Place with the tweezers the loading papers in the incision. Ensure yourself that the loading paper doesn’t touch the bottom of the gel tray and doesn’t protrude above the gel surface. If necessary adjust the loading paper by cutting with a pair of scissors.
 * 6) Fill the 2 buffer tanks with the running buffer.
 * 7) Apply the shammy bridges (see figure 1) and place a glass cover of the gel plate and on the bridges.
 * 8) Connect the power supply with the electrodes of the buffer tanks. The samples migrate from (-) to (+) pole.
 * 9) Place the safety cover over the electrophoresis equipment and switch on the power supply.
 * 10) Run for 4 – 6 hours at 0.3 kV and 7 mA, until the Hb A-band is 2 cm away from the upper bridge. If case a faster moving band is visible, end the run when this band is about 2 cm away from the bridge.

Staining of the gel

 * 1) Prepare the staining solution, under a flow cabinet.
 * 2) Switch off the power supply and the cooling system.
 * 3) Remove the loading papers from the gel, using tweezers.
 * 4) Place the cover glass back on the gel, separate the upper part of the gel from mthe main body by cutting the gel with a steel wire leaning on the edges of the plate and remove the upper part of the gel.
 * 5) Free the gel from the edges of the plate using the tip of a Pasteur pipet.
 * 6) Place the plate in a staining tray and add the staining solution (use glows).
 * 7) Remove the staining solution after about 20 minutes (use glows).
 * 8) Rinse once with 200 ml of 50% methanol dry the starch surface with tissue paper and make a picture with the digital camera (use glows).
 * 9) Keep the gel in 200 ml 50% methanol. Stained starch electrophoresis gels can be kept for over a one month and subsequently discarded as chemical waste.

Troubleshooting
New batches of starch may require an adjustment of the concentration. In case the gel

stays too soft increase the concentration. The amount of starch needed, may go from

10 to 12 g for 100 ml.