Strategiesfor

Protein PurificationPresented by:

Sourav Sharma2nd Year, B.Tech, Biomedical Engineering,

Netaji Subhash Engineering College, Kolkata.

University Roll No: 10903113019

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

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.

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.

Protein Properties (Physical)

Shape

Sizes

Density

Charge

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

Types of Centrifuge based on RPM

• Micro centrifuges12,000–13,000 rpm

• High Speed Centrifuges around 30,000 rpm

• Ultracentrifuges excess of 70,000 rpm

Types of Centrifuge based on RPM

Differential centrifugation

Equilibrium density-gradient centrifugation

SDS PAGE

• Separates Proteins from DNA, Lipids and Polysaccharides

• Separates Proteins based on their sizes

A B C D E F G

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.

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.

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)

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.

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.

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.

https://sharmasourav.wordpress.com/

End of Presentation

https://www.slideshare.net/secret/MXx0rt7XAFR22V

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