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Polyacrylamide gel electrophoresis

Procedure

Assembling the plate

  1. After cleaning the plate with ethanol 70%, assemble all the glass plate by placing the longer glass plate down first followed by the shorter glass plate on top of it. (Check with water if there is any leaking)

  2. Clamp them carefully and properly into the casting frame. On the same time, make sure that the bottom ends of the glass plates are properly aligned. Next, plate it on casting stand.

     

     

     

     

     

     

     

     

     

Assembling the plate

Casting the gel

  1. Generally, the gel consist pf acrylamide, bisacrylamide, the optional denaturant (SDS) and a buffer with an adjusted pH. Firstly, combine all reagents to prepare the separating gel solution. The acrylamide concentration of the gel can be varied, generally from 5% to 25%, the lower percentage gel more suitable for resolving high molecular weight molecules.

  2. Add a source of free radicals and stabilizer such as ammonium per sulfate and TEMED to the monomer solution (just before pouring) and mix well by swirling gently.

  3. Allow the gel to polymerize for 20-30minutes between two glass plates in a gel caster, with a comb inserted at the top in order to create the sample wells.

 

Casting the gel in a proper way

4. Prepare the stacking gel. The stacking gel is the upper gel for proteins to concentrate before entering the separating gel. It is important for proteins to concentrated at the interface between separating and stacking gel, thus they will enter the separating gel at the same time to ensure the comparable migration pattern.

5. Clean comb with ethanol 70% before place it between the two glasses.

6. Solution is mixed gently and add in between the two glasses followed by polymerization of the gel.

7. Remove the comb after the gel is polymerized for about 15-30 minutes and clean the comb with water.

8. Remove the comb after the gel is polymerized for about 15-30 minutes and clean the comb with water.

 

Preparation of the sample

  1. Sample is any material containing proteins or nucleic acids. The sample can be mixed with a chemical denaturant such as SDS.

  2. Besides to this, proteins can be briefly heated to near boiling to denatures the proteins.

    1. Boiling for 5-10 minutes. (Works for most proteins)

    2. 65°C for 10 minutes. 

    3.  37°C for 30 minutes.

 

Electrophoresis (Running the SDS- Gel)

  1. To assemble, take out the gels from the casting frame and clamp them in the gel apparatus followed by lock the plates in cassette.

  2. Place them in the gel running tank.

  3. Adding the buffer carefully in upper chamber but not to empty the wells.

  4. Remove the comb carefully.

  5. Insert the loading tip to a few mm from the well bottom and deliver the samples into the well. Rinse the syringe with distilled water after loading for a few times.

  6. Place the lid on top of the chamber. Make sure connections are correct (red with red and black with black).

  7. Set the voltage up to 180 V and run for 1 hour.

 

Staining the gel

For allowing the visualization of the separated proteins, the gel may be stained (for proteins, most commonly with Coomassie Brilliant Blue R-250; for nucleic acids, ethidium bromide; or for either,silver stain). Place the gel in the staining solution for 30 minutes. Remove the staining solution and replace by destaining solution when the bands are properly seen. The different species of biomolecules will appear as distinct bands within the gel after staining.

 

Calculate and determine the approximate molecular weight of the unknown sample by comparing them with the molecular weight ladders (markers).

 

 

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