Characterization of nucleic acids and protein by electrophoresis

1. Characterization of Nucleic Acids
and Protein by Electrophoresis
Apeksha Niraula
Junior Resident-II
Department of Biochemistry

2. Contents

3. Electrophoresis
Electrophoresis is a term that refers to the migration of
charged solutes or particles of any size in a liquid medium
under the influence of an electrical field.

4. Theory
Movement of a charged molecule subjected to an electric field is
represented by
V= Eq /f
Where, E= Electric field in volts/cm
q= Net Charge on the molecule
f= Frictional Coefficient ; which depends on the
mass and shape of the molecule
v= Velocity of the molecule

5. µ (mobility) = v/E
i.e
µ = q/F

6. Methods of Electrophoresis
 Major difference in methods Electrophoresis is the
type of supporting medium.
 Can be either cellulose or thin gels.

7. Polyacrylamide Gel Electrophoresis
 Gels are formed by polymerization of acrylamide.
 Polyacrylamide is prepared by the free radical
polymerization of acrylamide and the cross-linking agent
N,N’-methylene bis-acrylamide.
 Chemical polymerization is controlled by an initiator
catalyst system;
Ammonium Persulfate- N,N N’N’ Tetramethylene diamine
(TEMED).
 Photochemical Polymerisation may be initiated by
Riboflavin in the presence of Ultraviolet radiation(UV
Radiation).

9. Features of Polyacrylamide Gel:
 High resolving power for small and moderately sized proteins and
nucleic acids (upto 1x 106 Daltons).
 Acceptance of relatively large sample size.
 Minimal interactions of the migrating molecules with the matrix.
 Good physical stability of the matrix.

10.  Gels can be prepared of different pore sizes by changing the
concentration of crosslinking agents.
 Standard gel for protein separation – 7.5% Polyacrylamide.
 PAGE provides enhanced resolution of sample
components As separation is based on
molecular sieving and
Electrophoretic mobility.

11. PAGE can be done using either of two arrangements:
 Column
 Slab

15. Disadvantage of PAGE:
 Difficult and inconvenient to prepare polyacrylamide gels.
 Difficult to make gels of reproducible thickness and
composition.

16. Discontinuous Gel Electrophoresis
Three significant characteristics:
 Two Gel layers – Lower or Resoving Gel
- Upper/ Stacking Gel
 Buffers used to prepare the two gel layers are of different
ionic strengths and pH.
 Stacking Gel has a low acrylamide concentration
Its Pore sizes are larger.

17.  These three changes in the experimental conditions
cause the formation of highly concentrated bands of
sample in the stacking gel and greater resolution of the
sample components in the lower gel.
 Sample Concentration in the upper gel occurs in the
following manner :
 Sample is usually dissolved in Glycine-Chloride
Buffer (pH 8-9) before loading onto the gel.
 Voltage turned ON Buffer Ions( Glycinate and
Chloride) and Protein or Nucleic Acid sample move into
the Stacking Gel which has pH of 6.9.

18. H3N+CH2COO- H2NCH2COO- + H+
Upon Entry into the upper Gel
Equilibrium of equation 4.4 shifts into the left.
Increasing the concentration of zwitter ion
which has no net charge and hence no
electrophoretic mobility.

19.  Since most proteins and nucleic acids are anionic at pH
6.9 Replace Glycinate as mobile Ions.
 The relative ion mobilities in the Stacking Gel are
Chloride> Protein/Nucleic Acid > Glycinate.

20.  Sample tend to accumulate to form a thin concentrated band
sandwiched between the Chloride and Glycinate as they move
through the upper gel.
 When the ionic front reaches the lower gel with pH 8-9
buffer, the glycinate concentration increases and anionic
Glycine and Chloride carry most of the current.
Protein or Nucleic acid sample molecules now in a narrow
band Encounter both an increase in pH and decrease
in Pore size.

21.  Relative rate of movement of Anions in the Lower gel
Chloride > Glycinate > Protein or Nucleic Acid Sample.
 Each component has unique mass/charge ratio,discrete
size and shape Directly influence its mobility.
 Increase in pH Increase Electrophoretic Mobility
 Smaller Pore Size Decrease in Mobility.
 Yields Excellent Resolution method of choice for Analysis of
Protein/Nucleic Acid fragments.

23. Sodium Dodecyl Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
 Used for the measurement of the molecular weight of biological
molecules.
 If Protein Samples are treated So that they have a
Uniform Charge
{Electrophoretic Mobility then depends primarily on
Size}.

24. Protein + SDS(1%)
SDS disrupts 20 30 and 40 structure to produce linear
polypeptide chains coated with negatively charged SDS
molecules.
Mercaptoethanol(0.1 M) assists in protein denaturation by
reducing all disulfide bonds.
Detergent binds to hydrophobic region of the
denatured protein in a constant ratio of 1.4 gm of SDS/gm
of protein.

25. The bound deteregent molecules carrying negative charges mask
the native charge of the protein.
Hence, Polypeptide chains of a constant charge /mass ratio and
uniform shape are produced.
 Two sets of standards are available; one for low molecular
weight (14,000-100,000) and one for high molecular weight
protein (45,000-2,00,000).
 Mobilities are measured and molecular weight determined
graphically.

26. Nucleic Acid Sequencing Gels
 Amino acid sequence of a protein is determined by
identifying amino acid residues.
 Sequence analysis of nucleic acids is based on the generation
of sets of DNA/RNA fragments with common ends and the
separation of these oligonucleotide fragments by PAGE.

27. Two methods have been developed for sequencing
nucleic acids:-
I. Partial Chemical degradation method of Maxam and Gilbert .
II. Chain Termination method developed by Sanger.
 Both sequencing methos result in nested sets of DNA or RNA
fragments that have one common end and chains varying in
length.

28.  Sequence gels of 6,8,12,and 20% Polyacrylamide are routinely
used.
 Gels of 20% may be used to sequence the first 50-100
nucleotides of a nucleic acid and lower percentage gels allow
sequencing out to 250 nucleotides.
 Denaturants such as Urea and Formamide are required
to prevent renaturng of the nucleic acid fragments during
electrophoresis.

29.  For detection, Nucleic Acid chains for sequencing must be
end labeled with 32P,35S or a fluorescent tag.
 32P and 35S labeled nucleic acids on gels are
detected by Autoradiography and those labeled with
fluorescent molecules are etected by Fluorimeter
scanning of the gels.

30. Agarose Gel Electrophoresis
 Standard method used to characterize DNA/RNA in the
range 200-50,000 kilobase pairs (kilobases).
 Uses Agarose (Sea-weed) ,a linear polymer of
galactopyranose derivatives.
 Agarose concentration of 0.3%-0.5% most effective for nucleic
acid separation.
 Like proteins,Nucleic acids migrate at a rate inversely
proportional to the logarithm of their molecular weights.

31. The DNA confirmation most frequently encountered are:
 Superhelical Circular (Form I)
 Nicked Circular (Form II)
 Linear (Form III)
 According to mobility (I>III>II)

32.  Two of many applications of Agarose gel Electrophoresis.
 Analysis of DNA fragments after digestion by Restriction
Endonuclease.
 Characterization of superhelical structure of DNA.

33. Pulsed Field Gel Electrophoresis (PFGE)

34. Isoelectric Focusing of Proteins

35. Two Dimensional Electrophoresis
 Capillary Electrophoresis

36. Immunoelectrophoresis

37. CLINICAL ASPECTS