Molecular Techniques and Methods

Starch Gel Electrophoresis of Proteins

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Starch gel has simple system. Starch and buffer solutions are only reagents required. The completely non-toxic character of starch must be contrasted with the toxicity of acrylamide monomer. Starch gels are prepared by heating and cooling a quantity of partially hydrolyzed starch in an appropriate buffer solution. A characteristic feature of starch gels is that they exhibit molecular sieving effects. Separation of proteins is achieved, therefore, not only on the basis of differences in charge, but also of differences in molecular size and shape.
The starch gels are prepared with total concentrations between 5 and 15% (w/v), but 10% is a good starting point for most separations. The thick layer (6 mm) starch gel is used where the availability of sample is not a critical factor. They have been widely used to study a variety of protein and enzyme polymorphisms. The thin layer (1 mm) starch gel is most suitable for the electrophoretic separation of small quantities of protein mixtures, as encountered in forensic analysis.




MATERIALS AND SOLUTIONS


Tank Buffer (pH 4.9)
Tank Buffer (pH 7.4)
Tank Buffer (pH 8.5)
Citric acid - 28.7 g
Maleic acid - 11.6 g
Diethylbarbituric acid - 2.4 g
NaOH - 10.7 g
Tris - 12.1 g
Sodium barbiturate - 4.4 g
H2O - 1 liter
H2O - 1 liter
H2O - 1 liter
Adjust pH w/ HCl
Adjust pH w/ NaOH
Adjust pH w/ NaOH



Gel Buffer (pH 5.0)
Gel Buffer (pH 7.4)
Gel Buffer (pH 8.6)
Succinic acid - 0.95 g
Citric acid - 0.8 g
Citric acid - 1.1 g
Tris - 1.2 g
Tris - 1.6 g
Tris - 9.2 g
H2O - 1 liter
H2O - 1 liter
H2O - 1 liter
Adjust pH w/ Acetic acid.
Adjust pH w/ NaOH.
Adjust pH w/ NaOH.



Glass Plates
25 cm (long) X 15 cm (wide) X 4 mm (thick)


Glass Edge Strips for Thick Layer Starch Gel
25 cm (long) X 5 mm (wide) X 3 mm (thick)
15 cm (long) X 5 mm (wide) X 3 mm (thick)


Glass Edge Strips for Thin Layer Starch Gel
25 cm (long) X 5 mm (wide) X 1 mm (thick)
15 cm (long) X 5 mm (wide) X 1 mm (thick)


Protein Staining Solution (110 ml)
Naphthalene black ------------------------------------- 10 g
Methanol ---------------------------------------------- 50 ml
Deionized H2O ---------------------------------------- 50 ml
Glacial acetic acid ------------------------------------- 10 ml


Destaining Solution (110 ml)
Methanol ---------------------------------------------- 50 ml
Deionized H2O ---------------------------------------- 50 ml
Glacial acetic acid ------------------------------------- 10 ml




PROCEDURES

Preparation of Starch Gel

1. Mix the 5 g dry starch with the 50 ml Gel Buffer (pH 5.0, 7.4, or 8.6) to form a lump free suspension.

2. Heat the suspension until it has thickened and become less viscous.

3. Degas by partial evacuation.

4. Pour on to a glass plate.

5. Cover the surface before setting has taken place.

6. Prepare the gel mold by sticking the 3-mm thick Glass Edge Strips to one of the glass plates.

7. Place a second set of Glass Edge Strips (3-mm thick) on top of the first.
These are held in place by smearing the joining surfaces with a non-silicone grease.
This will give a tray of 22 cm (long) X 12 cm (wide) X 6 mm (deep).

8. For a 10% gel, mix 20 g of the dry starch with 200 ml Gel Buffer (pH 5.0, 7.4, or 8.6).

9. Heat the suspension with continuous vigorous swirling and bring the solution to a boil.

10. Continue boiling for a few seconds and then degas the solution by applying a vacuum to the flask.

11. Place the mold on a horizontal surface, pour the hot starch solution into the center, and allow it to spread over the surface of the mold.

12. If air bubbles form or if the solution does not reach the edge, touch the solution at the point with a rubber-gloved finger.

13. Leave the plate for 30 minutes to allow the starch solution to cool and gel.
Do not disturb the gel during this period.

14. Transfer the gel plate to a moisture cabinet at 4oC and keep overnight.


Sample Applications

15. Make sample application slots in the gel across the width of the plate approximately 6 cm from the cathode (-) end. These can be made individually by inserting a piece of razor blade, approximately 1 cm wide, vertically into the gel.

16. Soak a piece of Whatman 3 MM filter paper (1 X 0.5 cm) in the protein sample solution (1-100 ug/ul).

17. Insert protein sample soaked Whatman paper into a sample slot.
Ensure the inserts reach the base glass plate.
The inserts absorb about 20 ul of protein sample solution (1-100 ug/ul).


Electrophoresis

18. Place the gel plate in an electrophoresis tank and apply wicks to both ends of the gel.
The wicks, consisting of several thicknesses of filter paper and cut to the width of the gel, should be soaked in Tank Buffer (pH 4.9, 7.4, or 8.5), and overlap the gel surface by 1-2 cm. They can be held in place by a plain glass plate (22 x 15 cm), which will also cover the gel and reduce evaporation.

19. The gels must be cooled during electrophoresis by placing the whole tank in a cold room at 4oC.

20. Electrophorese between 2 and 20 volts/cm with running times between 2 and 16 hours.
  • (It is difficult to generalize about the conditions of electrophoresis: time, voltage, and current are selected to give the best separation for the protein system being studied.)


  • 21. After electrophoresis, remove the top plate and wicks, lift the gel plate from the electrophoresis tank, and apply a suitable stain.

    22. For thin layer starch gel, apply stain directly (Go to step 26).
    For thick layer starch gel, slice the gel in half. To do this, remove the filter paper inserts and the second set of edge strips.

    23. Place a glass plate on the surface of the gel and draw a thin wire through the gel. During the cutting process, ensure that the wire is kept firmly pressed to the surfaces of the bottom edge strips.

    24. Remove the top glass plate and roll a piece of damp filter paper onto the gel surface. The upper half of the gel will adhere to the paper and can be peeled away from the lower half.

    25. The cut surfaces of each half can now be stained.


    Staining

    26. Stain the gel in the Portein Staining Solution for 2-5 minutes.

    27. Wash the gel in a large volume of the Destaining Solution for 5-10 hours.

    28. The gel is transformed into an opaque solid strong enough for convenient handling.

    29. The areas which contain protein remain a dark blue colour and contrast well with the white background.




    NOTES

  • Hydrolyzed starch suitable for electrophoresis can be prepared in the laboratory by warming granular potato starch in acidified acetone.


  • The starch solutions take between 5 and 30 minutes to gel, but it has been noticed on numerous occasions that superior resolution of protein bands is achieved if the gels are stored in a moisture cabinet overnight.


  • After preparation, the gels should appear clear or slightly opalescent and homogenous.


  • The sensitivity of the method is ultimately dependent on the staining procedure used. Samples that have a total protein concentration of between 1 and 100 mg/ml are generally used.




  • KIT INFORMATION




    REFERENCES

  • Gordon, A.H., 1975, Electrophoresis of proteins in polyacrylamide and starch gels, American Elsevier Publishing Company, Inc. New York.


  • Smithies, O., 1955, Zone electrophoresis in starch gels: group variations in the serum proteins of normal adults. Biochem. J. 61, 629-641.


  • Wraxall, B.G.D., and Culliford, B.J., 1968, A thin-layer starch gel method for enzyme typing of bloodstains. J. Forensic Sci. Soc. 8, 81-82.


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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