Electrophoresis and affinity chromatography are techniques used to separate and purify biomolecules. Electrophoresis separates molecules based on size and charge by applying an electric current through a gel, causing smaller molecules to migrate faster. Affinity chromatography uses a ligand attached to a solid support to selectively bind to target molecules based on specific interactions, allowing purification of the bound target molecules. Both techniques are widely used to purify proteins and nucleic acids.

1. ELECTROPHORESIS
& AFFINITY
CHROMATOGRAPHY
ABSTRACT
A Complete note regarding electrophoresis & affinity
chromatography technique. Chromatography is widely
used for purification and separation of biomolecules
and others molecules. Also, electrophoresis technique
is used to purifications of proteins and DNA.
gp.qa226@hotmail.com
INSTRUMENTAL METHODS OF ANALYSIS

2. Electrophoresis
Electrophoresis is the motion of dispersed particles relative to a fluid under the
influence of a uniform electric field. Thus it separates components of a mixture
based on their size amd/or charge.
Paper electrophoresis
Generally used to separate AAs or peptides of differing charge. As shown in the
figure, AAs and peptides will separate based on their charge with the most highly
charged species moving the furthest.

3. PAGE (PolyAcrylamide Gel Electrophoresis) -- Native Gel
It is used in clinical chemistry to separate proteins by charge and/or size (IEF
agarose, essentially size independent) and in biochemistry and molecular biology
to separate a mixed population of DNA and RNA fragments by length, to estimate
the size of DNA and RNA fragments or to separate proteins by charge. It is a
process which enables the sorting of molecules. Using an electric field, molecules
(such as DNA) can be made to move through a gel made of agar or
polyacrylamide. The electric field consists of a negative charge at one end which
pushes the molecules through the gel, and a positive charge at the other end that
pulls the molecules through the gel. The molecules being sorted are dispensed into
a well in the gel material. The gel is placed in an electrophoresis chamber, which is
then connected to a power source (see figure to the left). When the electric current
is applied, the larger molecules move more slowly through the gel while the
smaller molecules move faster. The different sized molecules form distinct bands
on the gel.
The term "gel" in this instance refers to the matrix used to contain, then separate
the target molecules. In most cases, the gel is a crosslinked polymer whose
composition and porosity is chosen based on the specific weight and composition
of the target to be analyzed. When separating proteins or small nucleic acids
(DNA, RNA, or oligonucleotides) the gel is usually composed of different
concentrations of acrylamide and a cross-linker, producing different sized mesh

4. networks of polyacrylamide. When separating larger nucleic acids (greater than a
few hundred bases), the preferred matrix is purified agarose. In both cases, the gel
forms a solid, yet porous matrix. Agarose is composed of long unbranched chains
of uncharged carbohydrate without cross links resulting in a gel with large pores
allowing for the separation of macromolecules and macromolecular complexes.
Electrophoresis" refers to the electromotive force (EMF) that is used to move the
molecules through the gel matrix. By placing the molecules in wells in the gel and
applying an electric field, the molecules will move through the matrix at different
rates, determined largely by their mass but also their charge and shape which
varies widely for proteins. Electrophoretic mobility of small molecules is greater
than the mobility of large molecules with the same charge density thus allowing
separation. To separate proteins or DNA generally the pH of the buffer and protein
mixture is high (~9) so that the proteins carry a net-negative charge. However,
because size, charge and shape all play a role in how a molecule will behave in a
native gel most scientists use a SDS-PAGE gel which is predictable.
2D Gel Electrophoresis
Two-dimensional gel electrophoresis (2D electrophoresis) is a form of gel
electrophoresis commonly used to analyze proteins in which mixtures of proteins
are separated by two properties in two dimensions on gels. As shown in the figure,
2D electrophoresis begins with an IEF gel (in a tube) which separates proteins
based on their pI. This is then laid on top of a SDS-PAGE gel (90 degrees from the
first separation). Because it is unlikely that two molecules will be similar in two
distinct properties, molecules are more effectively separated in 2D electrophoresis
than in 1D electrophoresis.

5. Affinity chromatography
Affinity chromatography is a separation method based on a specific binding
interaction between an immobilized ligand and its binding partner. Examples
include antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions. The
degree of purification can be quite high depending on the specificity of the
interaction and, consequently, it is generally the first step, if not the only step, in a
purification strategy.
Mechanism of Affinity Binding
A commonly used metaphor to illustrate affinity binding is the lock and key
analogy. A unique structure present on the surface of a protein is the key that will
only bind to the corresponding lock, a specific ligand on a chromatographic
support.

6. Affinity-tagged purification.
In two-step affinity-tagged protein purification, a protein is first purified by affinity
chromatography, then desalted. In some medium pressure chromatography
systems, such as the NGC medium pressure chromatography systems, these two
steps can be automated. In the first step, a recombinant protein mixture is passed
over a chromatography support containing a ligand that selectively binds proteins
that contain an affinity-tag sequence (typically His or GST). Contaminants are
washed away, and the bound protein is then eluted in pure form.
Affinity tags have different advantages. In immobilized metal affinity
chromatography (IMAC), His binds with good selectivity to Ni2+
or other transition
metals immobilized to the ligand; the tagged protein can be selectively eluted with
imidazole. proteins tagged with GST bind to glutathione as the ligand, and are
eluted with solutions of glutathione. Proteins with an enzymatically active GST
fusion tag can only be purified under native conditions. In contrast, polyhistidine-
tagged proteins may be purified under native or denaturing conditions.
During the second step of desalting, affinity-purified samples can simultaneously
undergo buffer exchange to remove salts in preparation for downstream
applications.

7. A number of desalting techniques, including size exclusion chromatography,
dialysis, and ultrafiltration, also allow buffer exchange. Desalting often includes
the removal not only of salt, but also of other foreign substances, such as
detergents, nucleotides, and lipids.
These events can be summarized into the following three major steps:
1. Preparation of Column
• The column is loaded with solid support such as sepharose, agarose,
cellulose etc.
• Ligand is selected according to the desired isolate.
• Spacer arm is attached between the ligand and solid support.
2. Loading of Sample
• Solution containing a mixture of substances is poured into the elution
column and allowed to run at a controlled rate.
3. Elution of Ligand-Molecule Complex
• Target substance is recovered by changing conditions to favor elution of
the bound molecules.
Applications of Affinity Chromatography
• Affinity chromatography is one of the most useful methods for the
separation and purification of specific products.
• It is essentially a sample purification technique, used primarily for
biological molecules such as proteins.
Its major application includes:
• Separation of mixture of compounds.
• Removal of impurities or in purification process.
• In enzyme assays
• Detection of substrates
• Investigation of binding sites of enzymes
• In in vitro antigen-antibody reactions

8. • Detection of Single Nuceotide polymorphisms and mutations in nucleic
acids
Advantages of Affinity Chromatography
• High specificity
• Target molecules can be obtained in a highly pure state
• Single step purification
• The matrix can be reused rapidly.
• The matrix is a solid, can be easily washed and dried.
• Give purified product with high yield.
• Affinity chromatography can also be used to remove specific
contaminants, such as proteases.