PROTEIN PURIFICATION

1. PROTEIN PURIFICATION
By
OLUWADARE AGUNBIADE

2. Protein Purification
• Protein purification is the process of isolating proteins
from a complex mixture of cells, tissues, or organisms. It
involves a series of steps that separate the desired protein
from contaminants, resulting in a highly purified protein
sample.
• Protein purification is a crucial technique in biochemistry
and molecular biology, allowing scientists to isolate
specific proteins from complex mixtures. This process is
akin to sifting through a bustling crowd to find a single
individual – it requires precision and a deep
understanding of the protein's unique characteristics

3. Why Purify Proteins?
• Functional Studies: To understand how a protein works, it's essential to study it
in isolation. Purification allows researchers to investigate a protein's enzymatic
activity, interactions with other molecules, and structural properties.
• Therapeutic Applications: Many drugs target specific proteins. Purifying these
proteins is crucial for developing and testing new medications.
• Industrial Uses: Proteins find applications in various industries, including food,
cosmetics, and biofuels. Purification ensures the quality and consistency of
these products.
• Biotechnology: Purified proteins are used as therapeutic agents, diagnostics,
and research tools.
• Pharmaceuticals: Protein-based drugs, such as insulin and antibodies, require
high purity.
• Basic research: Purified proteins enable scientists to study protein structure,
function, and interactions.

4. Steps in Protein Purification
• Cell disruption: Cells are broken open to release their
contents, including proteins.
• Centrifugation: Cell debris is separated from the protein-
containing supernatant.
• Filtration: Proteins are separated from smaller contaminants
using filters or dialysis membranes.
• Precipitation: Proteins are concentrated and purified using
precipitation methods, such as ammonium sulfate or heat
treatment.
• Chromatography: Proteins are separated based on their
properties, such as size, charge, or affinity.

5. Challenges and Considerations
Protein purification often involves a combination of these techniques to
achieve high purity. The choice of techniques depends on the specific
protein and the desired level of purity.
• Protein Stability: Maintaining the protein's native structure and activity
throughout the purification process can be challenging.
• Yield: Achieving high yields while maintaining purity is often a delicate
balance.
• Cost: Purification can be expensive, especially for large-scale
production.
• By carefully selecting and optimizing purification techniques, scientists
can isolate proteins in a pure and functional form, paving the way for
groundbreaking discoveries and innovative applications.

6. Cell Disruption and Homogenization
• Cell disruption and homogenization are critical steps in protein
purification, as they enable the release of proteins from cells and
tissues.
• Importance of Cell Disruption and Homogenization
• Release of proteins: Cell disruption and homogenization allow
proteins to be released from cells and tissues, making them accessible
for purification.
• Improved protein yield: Efficient cell disruption and homogenization
can improve protein yield by releasing proteins that may be trapped
within cells or tissues.
• Reduced contamination: Cell disruption and homogenization can help
reduce contamination by releasing proteins from cells and tissues,
making it easier to separate them from contaminants.

7. Methods of Cell Disruption and
Homogenization
• Mechanical Methods
• Grinding: Cells or tissues are ground into a fine powder using a mortar and pestle or a
grinder.
• Blending: Cells or tissues are blended using a blender or a homogenizer.
• Sonication: Cells or tissues are disrupted using high-frequency sound waves.
• French press: Cells or tissues are disrupted using a French press, which applies high
pressure to break open cells.
• Non-Mechanical Methods
• Enzymatic lysis: Cells are disrupted using enzymes that break down the cell membrane.
• Detergent lysis: Cells are disrupted using detergents that solubilize the cell membrane.
• Freeze-thaw lysis: Cells are disrupted by freezing and thawing, which causes the cell
membrane to rupture.
• Osmotic shock: Cells are disrupted by exposing them to a hypotonic solution, which
causes the cell membrane to rupture.

8. Factors to Consider
• Cell type: Different cell types may require different
methods of cell disruption and homogenization.
• Protein stability: Cell disruption and
homogenization methods should be chosen to
minimize protein degradation and denaturation.
• Scalability: Cell disruption and homogenization
methods should be scalable to accommodate large
quantities of cells or tissues.

9. Centrifugation
• Generally the first step after forming a crude extract is a simple filtration
or centrifugation to remove the large material.
• Centrifugation is a process that involves the use of the centrifugal force
for the sedimentation of mixtures with a centrifuge.
• This process is used to separate two immiscible liquids with more-dense
components of the mixture migrate away from the axis of the centrifuge,
while less-dense components of the mixture migrate towards the axis.
• Centrifugation alters the effective gravitational force on to tube/bottle so
as to more rapidly and completely cause the precipitate ("pellet") to
gather on the bottom of the tube. The remaining solution is properly
called the "supernatant".
• The supernatant liquid is quickly decanted from the tube/bottle without
disturbing the precipitate.

11. Protein Precipitation
• Protein precipitation is a widely used
technique in biochemistry and molecular
biology to concentrate and purify proteins
• . It involves the addition of a precipitant to a
protein solution, causing the protein to
aggregate and form a solid precipitate.

12. Methods of Protein Precipitation
• Salting Out: The addition of a high concentration of
salt, such as ammonium sulfate, to a protein solution
causes the protein to precipitate out of solution.
• Organic Solvent Precipitation: The addition of an
organic solvent, such as ethanol or acetone, to a
protein solution causes the protein to precipitate.
• Heat Precipitation: Some proteins can be precipitated
by heating the protein solution to a high temperature.
• Polyethylene Glycol (PEG) Precipitation: PEG is a non-
ionic polymer that can be used to precipitate proteins.

13. Factors Affecting Protein Precipitation
• pH: The pH of the protein solution can affect the
precipitation of the protein.
• Temperature: The temperature of the protein solution
can affect the precipitation of the protein.
• Salt Concentration: The concentration of salt in the
protein solution can affect the precipitation of the
protein.
• Protein Concentration: The concentration of protein
in the solution can affect the precipitation of the
protein.

14. Dialysis
• Dialysis is a procedure for exchanging the solvent around a
protein.
• In general the protein solution is placed inside a semi-
permeable membrane (dialysis bag) which is suspended in a
larger volume of buffered solution (see image to the right).
• The key to this procedure working is that the membrane
has to be permeable to water and ions, but not to your
protein of interest.
• Thus buffers & salts exchange until an equilibrium is
established between the inside & outside of the membrane.

16. Column Chromatography
• Column chromatography is a powerful tool for protein purification,
allowing for the separation of proteins based on their unique
properties.
• Column chromatography is a cornerstone technique in protein
purification, offering a versatile and effective means to isolate specific
proteins from complex mixtures. I
• Principles of Column Chromatography
• Column chromatography involves passing a protein mixture through a
stationary phase, which selectively interacts with the proteins.
• t leverages the differential interactions of proteins with a stationary
phase (the column matrix) to achieve separation.
• The proteins are then eluted from the column using a buffer, and the
separated proteins are collected.

17. Types of Column Chromatography:
• Size-Exclusion Chromatography (SEC): Separates proteins based on
their size, with smaller proteins eluting later.
• Ion-Exchange Chromatography (IEC): Separates proteins based on
their charge, with positively charged proteins binding to negatively
charged resins and vice versa.
• Affinity Chromatography: Separates proteins based on their specific
interactions with immobilized ligands, such as antibodies or enzymes.
• Hydrophobic Interaction Chromatography (HIC): Separates proteins
based on their hydrophobicity, with more hydrophobic proteins
binding to the stationary phase.
• Chromatofocusing: Separates proteins based on their isoelectric
point (pI), with proteins eluting in order of increasing pI.

18. Applications of Column Chromatography in
Protein Purification
• Purification of recombinant proteins: Column
chromatography is widely used to purify recombinant
proteins expressed in various host systems.
• Purification of antibodies: Column chromatography is used
to purify antibodies from serum or cell culture supernatants.
• Purification of enzymes: Column chromatography is used to
purify enzymes for various applications, including research,
diagnostics, and industrial processes.
• Purification of protein complexes: Column chromatography
can be used to purify protein complexes, such as protein-
protein or protein-DNA complexes.

19. Key Advantages of Column Chromatography
• High Resolution: Can separate proteins with
subtle differences in properties.
• Versatility: Various types of chromatography can
be used to target different protein characteristics.
• Scalability: Can be scaled up for large-scale
protein purification.
• Automation: Many steps can be automated for
increased efficiency and reproducibility.
•

23. High-Performance Liquid Chromatography
(HPLC)
• High-Performance Liquid Chromatography (HPLC) is a powerful analytical technique
used to separate, identify, and quantify the components of a mixture. It is widely
used in various fields, including pharmaceuticals, biotechnology, and environmental
monitoring.
Principles of HPLC
• HPLC is based on the principle of chromatography, where a mixture of compounds is
separated based on their interactions with a stationary phase and a mobile phase.
• Stationary Phase: A solid or liquid phase that is immobilized in a column.
• Mobile Phase: A liquid phase that flows through the column and carries the sample
components.
• Sample Injection: A small volume of the sample is injected into the column.
• Separation: The sample components interact with the stationary phase and are
separated based on their affinities.
• Detection: The separated components are detected using a detector, such as a UV
or mass spectrometer.

24. Types of HPLC
• Reversed-Phase HPLC (RP-HPLC): The most common type of HPLC,
where the stationary phase is non-polar and the mobile phase is polar.
• Normal-Phase HPLC (NP-HPLC): The stationary phase is polar and the
mobile phase is non-polar.
• Size-Exclusion Chromatography (SEC): Separates molecules based on
their size.
• Ion-Exchange Chromatography (IEC): Separates molecules based on
their charge.
Applications of HPLC
• Pharmaceuticals: Analysis of drugs and their metabolites.
• Biotechnology: Analysis of biomolecules, such as proteins and nucleic
acids.
• Environmental Monitoring: Analysis of pollutants and contaminants in
water and soil.
• Food Safety: Analysis of food contaminants and adulterants.

25. Electrophoresis
• Electrophoresis is a laboratory technique used to separate and
analyze mixtures of DNA, RNA, and proteins based on their size and
charge. It is a powerful tool in molecular biology, biochemistry, and
genetics.
• 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.
Principles of Electrophoresis
• Electrophoresis is based on the principle that charged molecules will
move through a matrix when an electric field is applied. The
movement of the molecules is influenced by their size, charge, and
the properties of the matrix.

26. Types of Electrophoresis
• Gel Electrophoresis: Uses a gel matrix, such as agarose or polyacrylamide,
to separate molecules.
• Capillary Electrophoresis: Uses a narrow capillary tube to separate
molecules.
• SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis):
Uses a gel matrix and SDS to separate proteins based on their size.
• Native PAGE (Polyacrylamide Gel Electrophoresis): Uses a gel matrix to
separate proteins based on their charge and size.
• Isoelectric Focusing (IEF): Uses a gel matrix and a pH gradient to separate
proteins based on their isoelectric point.
• Two-Dimensional Electrophoresis (2D-E): Uses two separate
electrophoresis steps to separate proteins based on their charge and size.

27. Applications of Electrophoresis
• DNA Analysis: Used to separate and analyze DNA
fragments, such as in DNA sequencing and genotyping.
• Protein Analysis: Used to separate and analyze proteins,
such as in protein identification and quantification.
• Gene Expression Analysis: Used to analyze gene
expression patterns, such as in microarray analysis.
• Forensic Analysis: Used to analyze DNA evidence in
forensic science.
• Clinical Diagnosis: Used to diagnose genetic disorders
and diseases, such as in genetic testing.

28. Advantages of Electrophoresis
• High Resolution: Can separate molecules with
high resolution.
• Sensitive: Can detect small amounts of
molecules.
• Flexible: Can be used to analyze a wide range
of molecules.
• Relatively Low Cost: Compared to other
molecular biology techniques.