Download The Theory of Electrophoresis

The Theory of Electrophoresis
Electrophoresis is the method of separating
biomolecules (protein & nucleic acids) using an
electric field. The factors which govern the
movement of the biomolecules through the
electric field include:
• charge,
• size,
• shape of the biomolecules
When charged molecules are placed in an
electric field, they migrate toward either the
positive or negative pole according to their
charge.
A porous gel acts as a sieve by slowing, or in
some cases, by completely stopping the
movement of biomolecules while allowing
smaller molecules to move freely.
Proteins and nucleic acids are
electrophoresed within a matrix or "gel".
Most commonly, the gel is made in the
shape of a thin slab, with wells for
loading the sample.
The gel is covered with an electrophoresis
buffer that allows an electric current to
flow through the gel.
Loading the gel
Nucleic Acid v. Protein Separation
Nucleic acids
• Possess a negative charge and migrate toward the
positive electrode so that size is the only factor that
determines the separation of these biomolecules.
Proteins
• Come in a wide variety of sizes (molecular weights)
and possess different charges based on their amino
acid composition so size & charge both determine
the separation of these biomolecules.
DNA Size Standards
1,353
872
603
310
194
2,072
1,500
900
600
23,130
9,416
6,557
4,361
2,322
2,027
300
100
X174
HaeIII
1,636
506/517
220/201
564
72
100 bp
DNA Ladder
12,216
5,090
Lambda
HindIII
1 kb DNA
Ladder
Protein Size Standards
A
190 kDa
120 kDa
85 kDa
60 kDa
50 kDa
40 kDa
25 kDa
20 kDa
B
-galactosidase (132 kDa)
BSA (78 kDa)
Carbonic anhydrase (36.4 kDa)
Soybean trypsin inhibitor 26.6 kDa)
15 kDa
10 kDa
Aprotinin (8.4 kDa)
Insulin (3.8 kDa)
A: Benchmark® Prestained Protein Ladder (Invitrogen)
B: Kaleidoscope Prestained Polypeptide Standards (Bio-Rad)