Biochemistry GMC Bhavnagar

Electrophoresis refers to the migration of charged solutes or particles of any size in a liquid medium under influence of an electrical field. Depending on the charge on the molecules, they move towards either anode or cathode.

Electrophoresis of positively charged particles (cations) is sometimes called cataphoresis, while electrophoresis of negatively charged particles (anions) is sometimes called anaphoresis.

Required reagent for protein electrophoresis

Hippurate buffer pH 8.8

1% Agarose gel

BPB(bromophenol blue) dye

Amido black stain

Methanol

5% glacial acetic acid

Sample Preparation

Steps of Serum protein electrophoresis

Gel preparation

1. Clean the glass slide with tape water and and dry it with tissue paper.
2. Cut a rectangle four borders from X-ray plate and Stick X-ray plate with feviquick to the periphery of glass slide so the thickness of gel is same as thickness of X-ray plate.
3. Prepare 1% Agarose and pour hot agarose gel to non siliconized rough glass slide.
4. Cover the gel with siliconized glass slide carefully without entrapping air bubbles.
5. Allow the gel to get solidify for 10 minutes.
6. After 10 minutes removes siliconized glass slide by sliding movement and take care so that gel does not broken down.
7. Gel is ready for sample application.
8. Immediately apply the sample to prevent excess drying.

Sample Applicator Preparation

Prepare comb from the stainless steel as shown in image. Attach the magnetic strip to the ruler. Stick the ruler with the platform by feviquick. Stick the Applicator to magnetic strip. Applicator can be move up and down.

Sample application

1. Put Rough non sliconized glass slide below the sample applicator.
2. Apply 4 micro liter sample over the sample applicator at the edge of applicator as shown in figure. so that excess sample will be removed on glass slide.
3. Lift up the sample applicator and wipe the back side of applicator so that backward running of sample can be prevented.
4. Replace the rough glass slide with glass slide having prepared gel.
5. Apply the sample near the one end of gel with the applicator and wait for 2 minutes to allow the samples to get completely absorbed within the gel.
6. Remove the applicator.

Running the samples in electrophoresis tank

1. Clean the electrophoresis tank with DI water and dry it with tissue paper before use.
2. Put the electrophoresis tank on a table having even & leveled surface.
3. Fill the cold buffer in the two compartment. Put the gel slide in electrophoresis chamber in such a way that sample applied should remain on cathodic side.
4. Connect the gel with buffer through strip of filter paper wick & cover the chamber with its lid.
5. Connect the electrode on the side of slide containing sample well to the cathode (-) & other electrode to the anode of the power supply.
6. Set voltage at 300 volt and & run for 10 minutes.
7. After 10 minutes,switch off the power supply & remove the slide.

Fixation

Put the slide in to methanol for 10 minutes for fixation so that proteins get denatured and remain at same position even after staining and destaining.

Drying of gel

Put the fixated glass slide in to hot air Owen at 50-60° C for 20 minutes.

Staining

Place the gel slide in to Amido black staining solution for 2 minutes.

Destaining

Remove the excess stain by putting the gel in to destaining solution for 15 minutes so that background becomes clear.

Interpretation of the result