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protein_electrophoresis [2023/06/15 17:32] – quality_managerprotein_electrophoresis [2025/01/08 16:15] (current) – external edit 127.0.0.1
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 [[quality_manual|QM home]] [[quality_manual|QM home]]
 ^Name^Unique ID^Edition^Date of Edition^ ^Name^Unique ID^Edition^Date of Edition^
-|Documentary procedure for protein electrophoresis|LSSTH/A/QM/01| 1 | 01-03-2023|   +|Documentary procedure for protein electrophoresis|LSSTH/A/QM/01| 1 | 15-06-2023|   
    
 ^Preparing authority^Approving authority^Review period^ ^Preparing authority^Approving authority^Review period^
 |All teaching staff|Quality Manager|1 year| |All teaching staff|Quality Manager|1 year|
 +
  
 =====principle===== =====principle=====
-Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. Electrophoresis of positively charged particles (cations) is sometimes called cataphoresis, while electrophoresis of negatively charged particles (anions) is sometimes called anaphoresis.+ 
 +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.
  
 {{ ::types-of-electrophoresis.png?nolink&200 |}} {{ ::types-of-electrophoresis.png?nolink&200 |}}
  
-Introduction 
-Electrophoresis refers to the migration of charged solutes or particles of any size in a liquid medium under influence of an electrical field. 
  
-Steps of Serum protein electrophoresis 
  
-Required reagent+**Required reagent for protein electrophoresis** 
 + 
 +[[Hippurate buffer pH 8.8]]
  
 [[1% Agarose gel]] [[1% Agarose gel]]
  
-[[TBE buffer pH 8.8]]+[[BPB(bromophenol blue) dye]] 
  
 [[Amido black stain]] [[Amido black stain]]
  
 Methanol Methanol
 +
 [[5% glacial acetic acid]] [[5% glacial acetic acid]]
-[[BPB(bromophenol blue) dye]] 
  
-Procedure+[[Sample Preparation]] 
 + 
 + 
 +**Steps of Serum protein electrophoresis** 
 + 
 +**Gel preparation** 
 + 
 +  - Clean the glass slide with tape water and and dry it with tissue paper.  
 +  - 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. 
 +  - Prepare 1% Agarose and pour hot agarose gel to non siliconized rough glass slide. 
 +  - Cover the gel with [[siliconized glass]] slide carefully without entrapping air bubbles. 
 +  - Allow the gel to get solidify for 10 minutes. 
 +  - After 10 minutes removes siliconized glass slide by sliding movement and take care so that gel does not broken down.  
 +  - Gel is ready for sample application.  
 +  - 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** 
 +  - Put Rough non sliconized glass slide below the sample applicator. 
 +  - 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. 
 +  - Lift up the sample applicator and wipe the back side of applicator so that backward running of sample can be prevented. 
 +  - Replace the rough glass slide with glass slide having prepared gel. 
 +  - 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. 
 +  - Remove the applicator. 
 +   
 +**Running the samples in electrophoresis tank** 
 + 
 +  - Clean the electrophoresis tank with DI water and dry it with tissue paper before use.  
 +  - Put the electrophoresis tank on a table having even & leveled surface.  
 +  - 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.  
 +  - Connect the gel with buffer through strip of filter paper wick & cover the chamber with its lid. 
 +  - Connect the electrode on the side of slide containing sample well to the cathode (-) & other electrode to the anode of the power supply. 
 +  - Set voltage at 300 volt and & run for 10 minutes. 
 +  - 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** 
 + 
  
-1. Gel preparation 
-Before applying the gel on the slide wash  slide on which gel will be applied and dry it with tissue roll.  
-Place slide on a levelled surface as shown in above figure. Stick microtap  
-Stick microtap twice to the border of glass slide. so the thickness of gel is same as thickness of microtap layer. 
  
-TBE Buffer preparation 
-1. Tris base- 10.9 gm 
-2. Boric acid- 3.92gm 
-3. EDTA- 190 mg 
-Dissolve all these substance in to 950 ml distilled water. Measure the pH of buffer. If it is less than 8.6 add NaOH crystal to make pH up to 8.6 
  
-**buffer preparation** 
  
  
-**gel preparation** 
  
-**sample preparation**