This slideshow shows the techniques used for Protein Separation for medicine, material or food.

1. Strategies
for
Protein Purification
Presented by:
Sourav Sharma
2nd Year, B.Tech, Biomedical Engineering,
Netaji Subhash Engineering College, Kolkata.
University Roll No: 10903113019

2. Monomer Polymer Function(s)
Nucleotides DNA & RNA Genetic Information
Amino Acids Proteins Chemical
Reactions(Enzymes),
Structure
Sugars Polysaccharides Energy & Structure
Fatty Acids Lipids Membrane Structure &
Stability

3. Food rich in Proteins
We break down proteins in the food, using the building
blocks from those proteins to make new protein that cells
need, like antibody, hormones, etc.

4. Protein Purification
Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture,
usually cells, tissues or whole organisms.
Separation steps usually exploit differences in protein size,
physio-chemical properties, binding affinity and biological
activity.
The methods used in protein purification can roughly
be divided into analytical and preparative methods.

5. Protein Properties (Physical)
 Shape
 Sizes
 Density
 Charge

6. Centrifugation
Centrifugation is a process which involves the use of
the Centrifugal Force for the sedimentation of heterogeneous
mixture with a centrifuge.
Based on RPM
 Micro centrifuges
 High-speed centrifuges
 Ultracentrifuges
Based on Process
 Differential centrifugation
 Equilibrium density-
gradient centrifugation
Types

7. Types of Centrifuge based on RPM
• Micro centrifuges12,000–13,000 rpm
• High Speed Centrifuges  around 30,000 rpm
• Ultracentrifuges  excess of 70,000 rpm

8. Types of Centrifuge based on RPM
 Differential centrifugation
 Equilibrium density-gradient centrifugation

9. SDS PAGE
• Separates Proteins from DNA, Lipids and Polysaccharides
• Separates Proteins based on their sizes
A B C D E F G

10. SDS-Polyacrylamide Gel Electrophoresis
 Electrophoresis in gels are efficient than in solutions.
 Proteins are exposed to the ionic detergent SDS.
 The pores in a polyacrylamide gel are quite small.
 The rate of movement is influenced by
 the gel’s pore size
 the strength of the electric field.

11. Chromatography
Affinity chromatography (AC)
Gel filtration (GF) or Size Exclusive Chromatography
Ion exchange chromatography (IEX)
Chromatofocusing (CF)
Types
Chromatography means "color writing"
There is a mobile phase and a stationary phase.

12. Affinity chromatography is a method of separating biochemical
mixtures based on a highly specific interaction such as that between
antigen and antibody, enzyme and substrate, or receptor and ligand.
Affinity Chromatography (AC)

13. Gel filtration (GF)
GF is simple to use and allows separation of
substances with differences in molecular size,
under mild conditions.
GF is a non-binding method
Chromatofocusing (CF)
Chromatofocusing separates proteins according to
differences in their isoelectric point (pI).
It is a powerful method and can resolve very small
differences in pI
A pH gradient is generated on the column as buffer
and chromatography medium interact.

14. Ion exchange chromatography (IEX)
 IEX separates proteins with differences in surface charge to give high-
resolution separation with high sample loading capacity.
 The separation is based on the reversible interaction between a charged
protein and an oppositely charged chromatography medium.
 Target proteins are concentrated during binding and collected in a purified,
concentrated form.

15. Present Scenario
 Protein purification is now performed in scales from
micrograms and milligrams in research laboratories to
kilograms and tones in industrial settings.
 The efficiency gained by the generic purification approaches
based on affinity tagging of the target protein has
revolutionized protein purification
 Some proteins may be very challenging to purify in an active and
stable form.
 The challenges in protein purification that still remain make it
worthwhile to gain solid knowledge about protein purification so that
the available methods can be selected and applied in an optimal way.

16. https://sharmasourav.wordpress.com/
End of Presentation
https://www.slideshare.net/secret/MXx0rt7XAFR22V

17. References
 Ion Exchange Chromatography & Chromatofocusing Principles and Methods- Online GE Handbook.
 Methods for Protein Analysis- MIT Tv
 Purifying Challenging Proteins-Principles and Methods: GE Healthcare Handbook
 Ion Exchange Chromatography- Theory and Principles,
https://www.youtube.com/watch?v=A8lTfhWdAwE
 Cell Fractionalism
,http://www.sumanasinc.com/webcontent/animations/content/cell_fractionation.swf