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Methodology for Amino Acids and Proteins

 
     
 

Electrophoresis in proteins on starch gels
 
Starch gel is another supporting medium used for electrophoresis, particularly as a horizontal gel for the fractionation of proteins. Partially hydrolyzed starch dissolved in any of a variety of buffer solutions is used for casting gel. The gel may be prepared at any concentration between 2% and 15% (w/v) although a 10% gel is ideal for most separations. The starch gel electrophoresis is used for analytical as well as preparative purposes by changing the thickness of the gel.
 


 
Principle
The separation is effected not only on the basis of difference in charge but also of differences in the molecular size and shape of the proteins since starch gets exhibit molecular sieving property.
 
Materials
Glass plates (22 x 12 x 0.4cm dimension). Glass edge strips 22 and 14cm long, 5mm wide and 1 and 3mm thick.
Gel buffer (pH 8.6)
Tris-HCl                       9.2g
Citric acid                    1.05g
Water to                       1L
Hydrolyzed starch (electrophoresis grade)
Electrode Buffer (pH 7.9)
Boric acid                    18.6g
Sodium hydroxide         2g
Water to                       1L




Procedure
1.
Prepare the gel mold by sticking the 1mm thick glass edge strips to one of the glass plates to give a shallow tray 21cm long, 14cm wide and 1mm deep. Place the mold on a horizontal surface.
2.
To prepare a 10% gel for analytical purpose, mix 4g of the dry starch with 40mL gel buffer in a 200mL conical flask to a fine suspension.
3.
Heat the suspension while gently swirling the flask, and bring the starch solution to a boil. The solution attains a clear transparent form.
4.
Degas the solution by applying vacuum to the flask for 5-10sec. Release the vacuum carefully to avoid any splashing of the solution.
5.
Pour the hot starch solution into the centre and allow it to spread over the plate to form an even layer.
6.
Leave the plate for 10min or longer to allow the starch solution to cool and gel. Transfer the gel plate and maintain in a moisture cabinet at 4°C, preferably overnight, before use.
7.
Make slots in the gel across the width of the plate approximately 6cm from the cathode end for sample application. This is easily achieved by using a comb. Press the comb into the gel and remove it carefully.
8.
Soak pieces of 1cm long cotton thread in the sample solution and insert into the slot in such a way that the thread touches the base glass plate and not above the gel level. By this way, approximately 2-3mL of sample solution is applied. To load 15-20mL of sample solution a piece of 3mm filter paper (1 x 0.5cm) is used instead of cotton thread.
9.
Place the gel plate in an electrophoresis tank and apply wicks to both ends of the gel. The wicks (several thickness of filter paper) cut to the width of the gel should be soaked in the electrode buffer, and placed overlapping the gel surface by 1-2cm. Place a glass plate over the wicks and gel to hold them as well as to reduce evaporation from the gel.
10.
Run the gel at 5-20V/cm. the running time may be between 2 and 15h depending upon the voltage applied. The electrophoresis should however be conducted in a cold room (4-8°C).
11.
56yhAfter the run, remove the top plate and wicks, lift the gel plate from electrophoresis apparatus and apply a suitable stain.
12.
For protein pattern, the gel may be stained with coomassie brilliant blue R 250 as described under SDS-PAGE. However, the starch gel electrophoresis of proteins is mainly used for studying a group of related proteins and therefore specifically stained as for isoenzymes.
 
 
Notes
1.
Hydrolyzed starch suitable for electrophoresis may be prepared by warming potato starch in acidified acetone.
2.
There are number of buffer systems used for starch gel electrophoresis. As the separation of a particular group of proteins depends upon the pH, ionic strength, buffer constituents etc., the buffer system is carefully chosen.
3.
Prior to use, check that the gel does not contain any undissolved starch, bubbles, bacterial growth etc.
4.
The sensitivity of the method depends upon the staining procedure used. Samples that have a total protein concentration of between 1 and 100mg/mL are generally used.
 
 
References
1. Smithies, O (1955) Biochem J 61 629.

 
     
 
 
     




     
 
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