This document discusses various methods for purifying proteins, including precipitation, size exclusion chromatography, ion exchange chromatography, affinity chromatography, electrophoresis, immunoprecipitation, and high performance liquid chromatography. The goal of protein purification is to isolate a single target protein from a complex mixture for characterization, analysis, or commercial use. Key steps exploit differences in protein size, charge, binding affinity and other properties to progressively separate the target protein from all others in the sample.

1. PROTEIN PURIFICATION
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2. Protein purification
• It is a series of processes to
isolate one or a few protein
from a complex mixture,
usually cells, tissues or whole
organisms.
• Protein purification is
imprortant for the
characterization of the function,
structure and interactions of the
protein of interest.
• The purification process may
separate the protein and non-
protein parts of the mixture, and
finally separate the desired
protein from all other proteins.

3. CONT..
• Separation of one protein from all others is
typically
• Separation steps usually exploit differences in
protein size, physico-chemical properties,
binding affinity and biological activity. The
pure result may be termed protein isolate

4. PREPARATIVE PURIFICATION
• Preparative purifications aim to produce a
relatively large quantity of purified proteins for
subsequent use
• . Examples include the preparation of
commercial products such
as enzymes (e.g.lactase), nutritional proteins
(e.g. soy protein isolate), and
certain biopharmaceuticals (e.g. insulin).

5. ANALYTICAL PURIFICATION
• Analytical purification produces a relatively
small amount of a protein for a variety of
research or analytical purposes, including
identification, quantification, and studies of
the protein's structure, post-translational
modifications and
function. Pepsin and urease were the first
proteins purified to the point that they could be
crystallized.

6. extraction
• If the protein of interest is not secreted by the
organism into the surrounding solution, the
first step of each purification process is the
disruption of i) repeated freezing and thawing,
ii) sonication, iii) homogenization iv)
homogenization by grinding, and v)
permeabilization by detergents

7. Precipitation
• In bulk protein purification, a common first
step to isolate proteins
is precipitation with ammonium
sulfate (NH4)2SO4.
• This is performed by adding increasing
amounts of ammonium sulfate and collecting
the different fractions of precipitate protein.
Ammonium sulfate can be removed
by dialysis.

8. Size exclusion chromatography
• Chromatography can be used to separate protein
in solution or denaturing conditions by using
porous gels. This technique is known as size
exclusion chromatography.
• The principle is that smaller molecules have to
traverse a larger volume in a porous matrix.
• Consequentially, proteins of a certain range in
size will require a variable volume of eluent
(solvent) before being collected at the other end
of the column of gel.

9. SIZE EXCLUSIVE

10. Ion exchange chromatography
• ion exchange chromatography separates
compounds according to the nature and degree
of their ionic charge.
• The column to be used is selected according to
its type and strength of charge.
• Anion exchange resins have a positive charge
and are used to retain and separate negatively
charged compounds (anions), while cation
exchange resins have a negative charge and are
used to separate positively charged molecules
(cations).

11. Free-floe electrophoresis
• Free-flow electrophoresis (FFE) is a carrier-
free electrophoresis technique that allows
preparative protein separation in a laminar
buffer stream by using an orthogonal electric
field.
• By making use of a pH-gradient, that can for
example be induced by ampholytes, this
technique allows to separate protein
isoforms up to a resolution of < 0.02 delta-pI.

12. Affinity chromatography
• Affinity Chromatography is a separation
technique based upon molecular conformation,
which frequently utilizes application specific
resins.
• These resins have ligands attached to their
surfaces which are specific for the compounds
to be separated. Most frequently, these ligands
function in a fashion similar to that of
antibody-antigen interactions.

13. cont
• This "lock and key" fit between the ligand and
its target compound makes it highly specific,
frequently generating a single peak, while all
else in the sample is unretained.
• Many membrane proteins
are glycoproteins and can be purified by lectin
affinity chromatography.

14. Purification of the tagged protein
• Another way to tag proteins is to engineer
an antigen peptide tag onto the protein, and
then purify the protein on a column or by
incubating with a loose resin that is coated
with an immobilized antibody.
• This particular procedure is known
asimmunoprecipitation. Immunoprecipitation i
s quite capable of generating an extremely
specific interaction which usually results in
binding only the desired protein. site between
the tag and the protein.

15. Cont..
• The purified tagged proteins can then easily be
separated from the other proteins in solution
and later eluted back into clean solution.
• When the tags are not needed anymore, they
can be cleaved off by a protease. This often
involves engineering aprotease cleavage site
between t

16. HPLC
• High performance liquid chromatography or
high pressure liquid chromatography is a form
of chromatography applying high pressure to
drive the solutes through the column faster.
This means that the diffusion is limited and the
resolution is improved.
• The most common form is "reversed phase"
HPLC, where the column material
is hydrophobic. The proteins are eluted by
a gradient of increasing amounts of an organic
solvent, such as acetonitrile.

17. HPLC

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