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  Section: Plant Lab Protocols
 
 
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Methodology for Amino Acids and Proteins

 
     
 
Isoelectric focusing
 
Isoelectric focusing (IEF) is an electrophoretic method for the separation of proteins according to their isoelectric points (pI). It is reproducible, sensitive and highly useful to resolve closely related proteins which may not be so well separated by other techniques.
 
 


Principle
Analytical IEF is carried out in a thin layer of polyacrylamide gel containing a large series of carrier ampholytes. When a potential difference is applied across the gel, the carrier ampholytes align themselves in order of increasing pI from the anode to the cathode, thus producing a uniform pH gradient across the gel. Under the influence of the electric field each protein migrates to the region in the gradient where the pH corresponds to its pI. The protein is electrically neutral at its pI and where it gets focused in the gel. After focusing, the separated components are detected by appropriate staining.
 
 
Materials
Acrylamide, bisacrylamide and sucrose, all analytical grade.
Riboflavin solution, 1mg/10mL. store refrigerated in brown bottle.
Carrier ampholytes of the suitable pH range (pH 3-10, pH5-7 or pH 4-8) store at 4°C.
Glass plates 20 x 15 x 0.4cm dimension.
Fixing Solution: 5g sulphosalicylic acid and 10g trichloroacetic acid (TCA) in 90mL distilled water.
Destaining Solution: Methanol: acetic acid: water in the ratio 3:1:6 (v/v)

t;text-align:justify;line-height:normal;"> Staining Solution: 0.2% coomassie brilliant blue R250 in the destaining solution. Filter before use.
Wick Solutions: 1M phosphoric acid for anode and 1M NaOH for cathode.
Electrofocusing system.
 
 
Procedure
1.
Stick strips of insulating tape 1cm wide and approximately 0.20mm thick around the edge of a clean glass plate. This shall give a very shallow tray of 18 x 13 x 0.015cm in which a thin polyacrylamide gel is cast.
2.
Dissolve the following components completely
Acrylamide                           1.94g
Bisacrylamide                      0.06g
Sucrose                                 5.0g
Riboflavin solution              0.25mL
Water                                     40mL
3.
Add 2mL of carrier ampholyte solution of the appropriate pH range to the above mix. Ensure that all the components are uniformly mixed by gently swirling the flask until poured. The entire solution will be sufficient for six plates.
4.
Place a glass mold in large tray with the taped surface uppermost. Wipe clean the surface with an alcohol moistened tissue or remove any traces of grease.
5.
Transfer approximately 7mL of the above solution to one end of the glass mold.
6.
Place a clean plain glass plate (20 x 15cm) one of the short edges along the taped edge of the mold adjacent to the acryamide-ampholyte solution. Gradually lower the top plate and allow the solution to spread over the mold without entrapping any air bubbles. Press the top plate into firm contact with the taped edge of the bottom plate.
7.
Lift the complete mold and top plate out of the large tray. Remove any gel material at the edges and bottom of the mold plate.
8.
Photopolymerize the gel for at least 3h under white light or bright direct sunlight to provide sufficient UV light.
9.
After polymerization wipe the outside surfaces with a wet tissue to remove any solid material, otherwise it may affect cooling during the run.
10.
The plates may be stored for a month in the dark at 4°C. the removal of the top plate is easier when cooled at 4°C for at least overnight.
11.
Prior to sample application, remove the top plate carefully, inserting a scalpel blade between the two glass plates at the corner. The whole gel should stick either at the bottom or top plate for use. Occasionally, part of the gel will stick to the top plate and part to the bottom plate. In such cases, the gel has to be discarded.
12.
Absorb sample solutions (5-8mL) on 5 x 5mm pieces of Whatman No. 1 filter paper and lay these on the gel surface along the length of the plate at about 5cm from the cathode edge. The protein concentration between 0.05 and 0.15mg is generally sufficient.
13.
Place the gel plate on the cooling plate of the elctrofocusing apparatus through which water at 4-8°C is circulated.
14.
Apply electrode wicks (strips of Whatman 17 filter paper) to the anode and cathode edges of the gel. The anode and cathode wicks are uniformly soaked in 1M phosphoric acid and 1M NaOH respectively before being placed. (instead, 1% acetic acid and 1% ethanolamine may also be used as a wick solutions).
15.
Apply a potential difference of between 130 and 160 V/cm across the plate. Focusing takes 2-4h. focusing is complete when the same components of the same sample is placed on the cathode and anode sides in parallel lanes reached similar zones.
16.
When focusing is complete, disconnect the power supply, remove the electrode and wicks and lift the gel plate from the tank carefully.
17.
Place the gel plate in the fixing solution for 15-20min. transfer the plate to the destaining solution for 15min. then transfer the gel to staining solution for 30min; and finally to destaining solution for about 20min until the protein bands are clearly visible. The entire process is done at room temperature.
18.
To preserve the gel, first immerse the destained gel in destaining solution containing 10% glycerol for 30-60min. soak a cellophane sheet in the same solution for a few minutes and wrap it around the gel and supporting glass plate to avoid curling of the gel. Avoid trapping air. Let the wrapped gel dry in a well-ventilating oven at 50°C.
19.
Photograph the gel for a permanent record.
 
 
Notes
1.
Prefocusing of the plates prior to sample application for establishing pH gradient is preferable.
2.
The sample may preferably be applied at the cathode and where the denaturation of most proteins does not take place, although theoretically sample can be applied at any place, for focusing, however.
3.
Agarose is also used as a supporting medium for analytical IEF.
4.
Preformed gels for focusing are available commercially.
5.
Preparative electrofocusing is done using vertical column stabilized by density gradients with sucrose, glycerol and ficoll.
 
 
References
1. Aweleh, Z L, Williamson, A R and Askonas, B A (1968) Nature  219 66.
2. Wrigley, C W (1968) J Chromatogr 36 362.
3. Radola, B J and Grasslin D (1977) In: Electrofocusing and Isotachophoresis (Ed de Gruyter) Berlin and New York.
4. Ranjan Prasad, Chandrasekaran, P and Sadasivan, S (1986) Genet Agr 40 255.

 
     
 
 
     




     
 
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