In this presentation, I wrote about techniques which is used to separate Biomolecule, like, NATIVE PAGE, 2D PAGE, GEL ELECTROPHORESIS

1. Separation of
biomolecules by
1. NATIVE PAGE
2. 2D PAGE
3. AGAROSE GEL ELECTROPHORESIS

2. Gel Electrophoresis
 Gel electrophoresis is a technique commonly used in laboratories to
separate charged molecules like, DNA, RNA, proteins according to their
size.
 Charged molecule move through a gel when an electric current is passed
across it.
 An electric current is applied across the gel so that one end of the gel has
positive charge and other end of gel has negative charge.
 The movement of charged molecules is know as migration. Molecules
migrate towards opposite charge.
 The gel consist of permeable matrix, a bit like a sieve thorough molecules
travel when an electric current is passed across it.
 Small molecules migrate through the gel more quickly and therefore travel
further than large fragments that migrate slowly therefore will travel
shorter distance. As a result molecules are separated by size.

3. Agarose gel vs polyacrylamide gel
Agarose
1. A complex Polysaccharide derived
from sea weed.
2. Consist of many molecules.
3. Gels contain long chain of interlinked
sugar to form a mesh work.
4. Important in sepaartion of Much
larger DNA fragments.
5. Separate DNA about 50-20000 bp.
6. Have comparatively low resolving
power.
7. Typical concentration 0.5 to 2%.
8. Non toxic and easy to handle.
9. Non uniform pore size.
Polyacrylamide
1. Made by digestion of acrylonitrile by
nitrile hydratase.
2. Consist of one large molecule.
3. Gels are made up of chemical
crosslinking of acrylamide and Bis-
acrylamide, producing a molecular
sieve.
4. Important in separation of proteinsa
as well as small nucleic acid such as
oligonucleotide, miRNA, tRNA etc.
5. Separate DNA about 5-500 bp.
6. Have high resolving power.
7. Typical concentration 6-15%.
8. Contain a potent neurotoxin.

4. 1. NATIVE PAGE
Native Polyacrylamide Gel electrophoresis (PAGE) used is for
Studying the composition and structure of native proteins. Here
native means proteins are in properly folded state, not denatured.
 In Native PAGE Proteins are prepared in a non- reducing non-
denaturing Sample buffer, which maintains the protein
secondary structure and native charge density.
 Separation is based upon charge, size, and shape of proteins.
 Proteins remain enzymatically active after separation.
 Mobility depends on Proteins charge and hydrodynamic size.
 Useful for separation or purification of mixture of proteins.

5. • Blue Native PAGE
 BN- PAGE is a native PAGE technique where coomassie brilliant blue dye
provides the necessary charges to the protein complexes for the
electrophoretic separation.
 It permits a high resolution Separation of multi-protein Complexes under
native conditions.
 The electrophoretic mobility of a complex is determined by the negative
charge of the bound Coomassie Dye and the size and Shape of complex .

6. • Clear Native PAGE
 CN-PAGE separates acidic water –soluble and membrane proteins in a
Polyacrylamide gradient gel , and in many cases has lower resolution than
BN-PAGE.
 It uses no charged dye so the electrophoretic mobility of proteins is
related to intrinsic charge of proteins.
 The migration distance depends on the protein charge, it’s size and pore
size of the gel.
 CN-PAGE is milder than BN-PAGE so it can retain labile supramolecular
assemblies of membrane protein complex that are dissociated under the
condition of BN-PAGE.
 CN-PAGE used as a very efficient microscale separation technique for
FRET analyses.

7. • Quantative preparative native
continuous PAGE
 QPNC-PAGE is a Bioanalytical, high resolution and highly accurate
technique to separate proteins quantitatively by isoelectric point.
 This varient of gel electrophoresis is used by biologist to isolate active or
native Metalloproteins in biological samples and to resolve properly and
improperly folded metal cofactor containing proteins in complex protein
mixture.

8. 2. 2D PAGE
 2D PAGE is a powerful and widely used method for the analysis of
complex protein mixtures Extracted from cells, tissues or other biological
sample
 This technique separate proteins in two steps according to two
independent properties 1)
First –dimension is Isoelectric focusing (IEF) which seperates proteins
according to their Isoelectric point (pI). 2) Second –
dimension is SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) ,
which separates proteins according to their molecular weight(MW)
 This technique was first developed by O’Farrell and klose in 1975.

9. SAMPLE PREPARATION
 Must Select appropriate method to get selected proteins from cellular
compartment of interest.
 Must break all non-covalent Protein-protein, protein-DNA, protein-lipid
interactions, disrupts S-S bonds.
 Must remove substances that might interfere with separation process such
as salts, polar detergents(SDS), lipids, polysaccharide, nucleic acids
 Must prevent proteolysis, accidental Phosphorylation, oxidation, cleavage,
Deamidation.
 Must try to keep proteins soluble during both phases of electrophoresis
process.
 Protein solubilization -
8M Urea (neutral Chaotrope) -
4% CHAPS (Zwitterionic detergent) -
5-20 mM DTT (to reduce Disulphide) -
2-20 mM tris base(for buffering) -
Carrier ampholytes or IPG buffer (up to 2% v/v) to enhance protein
solubility and reduce charge-charge interractions.

10. 1. Isoelectric focusing (IEF) First Dimension
Isoeletric point(pI) : PH at which a protein has a neutral charge, loss or
gain of protons in a PH gradient (In a PH below their pI proteins carry a
net positive charge and in a PH above their pI, they carry a net negative
charge.
1. In IEF , proteins are separated by electrophoresis in a PH gradient
based on their isoelectric point.
2. A PH gradient is generated in gel and an electric field is applied
across the gel .
3. At all PH other than isoelectric point , protein will be charged .
4. At it’s isoelectric point, since the protein molecule carry no net
charge it accumulates or focuses into sharp band.
5. Proteins applied in the first dimension will move along the gel and
will accumulate isoelectric point.

13. IMMOBILIZED PH GRADIENT AND IEF RUN
 Immobilized PH gradients are used for IEF because the fixed PH gradient
remain stable over extended run times over high voltages.
 This technique has high resolution,great reproducibility and allow high
protein load.
 Isoelectric focusing is run in the same solution that are used to extract or
Solublize the proteins.
 The IPG strips with the protein sample must be rehydrated in the
rehydration/sample buffer during which proteins samples are loaded into
the strips.
 After the run in IEF cell , proteins Focus as band on the strip according to
their isoelectric points.

14. 2nd dimension:SDS PAGE
1. SDS-PAGE is an electrophoretic method for Separating polypeptides
according to their molecular weights. This technique is performed in
polyacrylamide gels containing sodium dodecyl sulfate(SDS).
2. SDS is an anionic detergent which denatures the protein by breaking
disulfide bonds, SDS masks the charge of the proteins themselves and
form anionic complex have a roughly constant negative charge per unit
mass.
3. Besides SDS a reducing agent such as Dithiothreitol (DTT) is also added
to break any disulfide bonds present in proteins.
4. When proteins are treated with both SDS and a reducing agent, the
degree of electrophoretic separation within a polyacrylamide gel depends
largely on the molecular weight of the protein.
5. SDS linearizes the protein so that they may be separated strictly by
molecular weight.

15. SDS Run
 The Equilibrated IPG strip is placed on the top of SDS-PAGE gel which is
submerged in a suitable buffer and sealed in place with agarose gel.
 An electric current is applied across the gel , causing the negatively
charged proteins to move out of the gel and migrate across the gel.
 the proteins separate according to their sizes and therefore by their
molecular weight.
 It is common to run marker proteins of known molecular weight in a
separate lane in the gel, in order to calibrate the gel and determine the
weight of unknown proteins by comparing distance travelled relative to
protein.

17. Visualization
1. After electrophoresis the gel is stained to visualize
the separated proteins.
2. Commonly used stains are Coomassie brilliant blue,
Sypro ruby or silver stain different protein will
appear as distinct spot Within gel.
3. In silver stain, silver Colloid is applied to gel. Silver
binds to cysteine groups within the protein. The silver
is darkened by exposure to ultra-violet light. The
amount of silver can be related to darkness, and
therefore the amount of protein at a given location
on the gel.
4. Silver staining 100X more sensitive than coomassie
brilliant blue stain

18. Advantages of 2D PAGE
1. 2D-PAGE can accurately analyze thousands of protein in single run.
2. High- resolution:This technology resolves proteins according to both pI
and molecular mass and enables the characterization of proteins with
post-translational modifications that affect their charge state.
3. 2D-PAGE is also used to study differential expression of proteins
between cell types.
4. The proteins can be separated in pure form from the resultant spots.

19. Disadvantages of 2D PAGE
1. This technique include a large amount of sample handling.
2. Limited reproducibility, and a smaller dynamic range than some other
separation methods.
3. Difficulty in separation of hydrophobic proteins.
4. It is also not automated for high throughput analysis.
5. Certain proteins are difficult for 2D-PAGE to separate, including those that
are in low abundance, acidic, basic, very large, very small.

20. Application of 2D-PAGE
 The protein can be separated in pure form from spots which can be
quantified and also analyzed by MS (Mass spectrometry)
 Host cell protein analysis
 Quality control of proteins.
 Posttranslational modification studies.
 To study proteomics.
 Provides important information correlating the absence or presence of
individual protein characteristics of specific clinical conditions.

21. 3.AGAROSE GEL ELECTROPHORESIS
1. Agarose gel electrophoresis is a method of gel electrophoresis used in
biochemistry, molecular biology, genetics, and clinical chemistry to
separate a mixed population of macromolecules such as DNA, RNA, or
proteins in a matrix of agarose.
2. Agarose is a natural polymer extracted from seaweed that forms a gel
matrix by hydrogen-bonding when heated in buffer and allowed to cool.
3. It is the most popular medium for the separation of moderate and large
sized nucleic acid and have a wide range of separation.

22. Principle Of AGE
 Gel electrophoresis separate DNA fragments by size in solid support
medium such as agarose gel.
 Sample DNA is Pippetted into the sample well, followed by the application
of an electric current which causes the negatively charged DNA to migrate
(electrophorse) towards the anodal, positive(+) end.
 The rate of migration is proportional to size, smaller the fragments move
more quickly and wind up at the bottom of the gel.
 DNA is visualized by including in the gel an intercalating dye, Ethidium
bromide , DNA fragments take up the dye as they migrate through the
gel, illumination with ultraviolet light causes the intercalated dye to
fluorescence.
 A larger fragment fluorescence more intensely.

23. REQUIREMENTS
1. An Electrophoresis Chamber and power supply.
2. Gel casting trays- the open ends of trays are closed with tape while gel
is being cast.
3. Sample comb- around whIch medium is poured to form sample wells in
the gel.
4. Electrophoresis Buffer – Usually Tris- acetate-EDTA(TAE) or Tris-borate-
EDTA(TBE).
5. Loading buffer-Which containing dense(glycerol) to allow the sample to
fall into the sample wall.
6. Staining- By ethidium bromide.
7. Transilluminator-(An ultraviolet light box) Which used to visualize stained
DNA in gels.

24. Concentration of Agarose gel
 The percentage of agarose used depends upon the size of the fragments
to be resolved.
 The concentration of agarose is reffered to as a percentage of agarose to
volume of buffer (w/v) and agarose gels are normally in the range of
0.2%to 3%.
 The lower the concentration of agarose the faster the DNA fragments
migrate.
 In general if the aim is to separate large DNA fragments a low
concentration of agarose should be used, and if the aim is to separate
small DNA fragments high concentration of agarose is recommended.

25. STEPS
1. To prepare gel, agarose powder is mixed with electrophoresis buffer to the
desired concentration and heated in micowave oven to melt it.
2. Ethidium bromide is added to gel (final concentration 0.5 ug/ml) to facilitate
visualization of DNA after electrophoresis.
3. After cooling the solution,It is poured into a casting tray containing sample
comb and allowed to solidify at room temperature.
4. After the gel has solidified, the comb Is removed , taking care not to rip the
bottom of the wells.
5. The gel in plastic tray is Inserted horizontally into the electrophoresis
chamber and is covered with buffer.
6. Sample containing DNA mixed with loading buffer are then pippetted into
the sample wells, the lid and power loads are placed on the approaches, and a
current is applied.
7. The current flow can be confirmed by observing bubbles coming off the
electrodes.
8. DNA will migrate towards the positive electrode.
9. The distance DNA has migrated in the gel can be judged by visually
monitoring migration of tracking dyes.

27. Application of Agarose gel
electrophoresis
 Estimation of the size of DNA.
 Analysis of PCR products e.g in molecular genetic diagnosis.
 Separation of restricted genomic DNA prior to southern analysis,or of RNA
prior to Northern analysis.
 Agarose gel electrophoresis is commonly used to resolve circular DNA with
different supercoiling topology, and to resolve fragments differ due to
DNA synthesis.
 In addition to providing an excellent medium for fragment size analyses,
agarose gels allow purification of DNA fragments.

28. Advantages of agarose gel
electrophoresis
1. For most applications only a single-component agarose is needed and no
Polymerization catalyst are required, therefore agarose gel are simple and
rapid to prepare.
2. The gel is easily poured, doesn’t denature the samples.
3. The sample can also easily recovered.

29. Disadvantage of agarsoe gel
electrophoresis
 The gels can melt during electrophoresis.
 The buffer can become exhausted.
 Different forms of genetic material may run in unpredictable forms.

30. Name:Md Amim Akram
Faculty Roll no :18ZYB012
Enrollment No :GI0081
Bsc(Hons) zoology(mains) 5th Semester
Thank you
Prepared by