Electrophoresis

Electrophoresis

1. Defintions

   • Electrophoresis: Migration of ions in an electric field
     • Velocity is directly proportional to charge and inversely proportional to size and shape.
   • Relative Mobility (Rf)= How far your protein band goes compared to something fast (dye)
     • Plotting Rf vs Molecular Mass gives standard curve

2. Protein Gel Electrophoresis

   • Proteins have inherent charge differences but may also be coated with charged particles.
   • A gel is used to slow down movement, and prevent diffusion of the proteins and create seperation
   • Running the Gel
     • Samples are loaded into wells
       • Ladder added (important to determine sizes)
         • Power turned on (250V until blue dye reaches bottom)
   • Visualizing the Gel
     • Apparatus is dissembled
       • Plastic/Glass Plates are Removed
         • Gel is immersed in stain to show banding pattern
           • Coomassie Blue Staining
             • Stain 10 minutes, soak in buffer 10 minutes
           • Silver Nitrate
             • Staining for more sensitive applications
               • More complicated and time consuming
           • Fluorescent Dye Staining
             • Sensitive but requires specialized equipment

3. SDS Page

   • Sodium Dodecyl Sulfate Polyacrylamide Gel
     • SDS= A detergent that will denature proteins and give them a negative charge.
     • Acrylamide= A plastic monomer, used industrially that can be chained together.
     • Acrylamide Gel Ingredients
       • Acrylamide: Forms Chains
       • Bis- acrylamide: Branch chains/ crosslinking
       • Ammonium Persulfate +TEMED: Form free radicals to catalyze polymerization
       • + A suitable buffer salt solution
   • Procedure
     • Protein samples are boiled for 5 minutes in 5x loading buffer
       • 10% SDS, 20% Glycerol, 0.2M Tris-HCL pH 6.8, 0.05% bromophenol blue, 10mM B-mercaptoethanol
     • Gel is cast by mixing acrylamide, bisacrylamide APS, TEMED, SDS and buffer.
       • Poured between glass or plastic plates
         • Oxygen inhibits polymerization
         • Comb to form wells added before gel solidifies
   • Based on a single criteria= Size

4. Problems with Band Resolution

   • The height of a well can produce a vertically diffuse band
     • Options
       • Use less volume loaded per sample
       • Concentration Sample
       • Run a longer gel
       • Run a stacking gel

5. Stacking Gels

   • Top (Stacking): 5% Acrylamide, pH 6.8 w/ Tris-HCL
   • Bottom (running): 12% acrylamide, pH 6.8 w/ Tris-HCL
   • Movement
     • Stacking Portion
       • Cl- speed
         • Highly negatively charged, small, pH independent : Very fast
       • Glycine- Speed
         • pH=8.3, enters the gel: Slow
       • Protein- Speed
         • Not as fast as Cl-, but faster than glycine
     • Running Portion
       • Cl Speed
         • Still Fast
       • Glycine Speed
         • Fast
       • Protein- Speed
         • Gel is thicker
           • Small= Fast
           • Large= Slow

6. Urea Denaturing Gels

   • Heating and running samples in a gel made with 6M urea allows for proteins to denature but retain their native charge
   • Proteins run in this system will have their mobility affected by size and charge
     • Run at a specific pH to control protein charges

7. Native (Non-denaturing) Gels

   • Mobility of proteins depends on size, charge, and conformation
     • Runs slower than SDS page, and charge dependant pH limits samples
       • Useful for blood testing

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