This document discusses strategies for isolating, purifying, and characterizing proteins. It describes various methods for extracting proteins from tissues or cells, such as cryogenic grinding, ultrasound homogenization, and lysis buffers. Purification techniques are then outlined, including precipitation, size exclusion chromatography, and isoelectric focusing. Finally, methods for identifying purified proteins are summarized, like mass spectrometry, N-terminal sequencing, and analyzing protein structure using techniques like circular dichroism spectroscopy and X-ray crystallography.

1. ISOLATION, PURIFICATION
AND CHARACTERISATION
OF PROTEINS
BY:
SAUMYA PANDEY
113BT0579

2. SEPARATION STRATEGIES
• Before a protein can be analysed , it must first be isolated in a pure state, i.e.,
purified .Protein purification means separating the protein of interest from other
proteins and components
1.Extraction
2.Precipitation
3.Purification
4.Concentration
5.Storage

3. PROTEIN ISOLATION
A protein can be originated from different sources like Physiological liquids, tissue,
cell lines and microbes in which protein usually resides or are expressed after
genetic manipulation.
• Protein Isolation: Methods for isolation
1. CYROGENIC GRINDING (Grinding in liquid nitrogen)
• Used for hard tissues and cells like roots, stems, but also for hard-walled cells like
some algae and cyanobacteria.
• Low temperature protects the proteins during grinding
• Time consuming (manual grinding) or requiring suitable machinery.
2. Ultrasound homogenisation
• Used for soft tissues like some leaves, and a post-treatment after grinding
• Does not require freezing and thus may avoid artefacts of freezing, but may cause
artefacts by heating of the sample.

4. 3.FRENCH PRESS
• Used for individual cells (plant cell culture, algae or bacteria) without or with soft walls.
• Does not require freezing and thus may avoid artefacts of freezing.
• Requires very expensive machinery.
4. Lysis buffer
• Used only for bacteria or animal cells
• May cause degradation
• No machinery needed.

5. PROTEIN PURIFICATION : AN OVERVIEW OF PRINCIPLES
Separation by expression site
• Selective use of tissues or organelles
• Separation of soluble from membrane proteins by centrifugation
• Separation by size
• Ultra filtration
• Size exclusion chromatography
• Preparative native gel electrophoresis
• Separation by charge
• exchange chromatography
• Isoelectric focusing (as chromatography, in solution or in gel electrophoresis)
• Separation by specific binding sites
• Metal affinity chromatography using natural or artificial metal binding sites
• Immuno-Chromatography using immobilized antibodies
• Magnetic separation using magnetically tagged antibodies

6. CHROMATOGRAPHY
• It is method to separate molecules according to their distribution between the mobile and
stationary phases. For protein separation SEC is most often used .
GEL FILTRATION
This method is also known as size exclusion chromatography as it separate proteins on the
basis of their molecular mass related to their size. The stationary phase is packed into the
column is a gel consisting of roughly spherical particles with pores of defined size. These
gels are prepared from the agarose , acrylamide and dextran polymers.
For the mobile phase a suitable solution is applied as the mobile phase that carries the
sample of proteins of different diameter through gel beads.
MECHANISM
SEC works by trapping smaller molecules in the pores of the adsorbent materials
adsorption ("stationary phases"). The larger molecules simply pass by the pores because
those molecules are too large to enter the pores. Larger molecules therefore flow through
the column more quickly than smaller molecules, that is, the smaller the molecule, the longer
the retention time.

7. • A small molecule that can penetrate every region of the stationary phase pore system can enter a total
volume equal to the sum of the entire pore volume and the inter particle volume. This small molecule will
elute late (after the molecule has penetrated all of the pore- and inter particle volume -- approximately 80%
of the column volume). At the other extreme, a very large molecule that cannot penetrate any the smaller
pores can enter only the inter particle volume (~35% of the column volume) and will elute earlier when this
volume of mobile phase has passed through the column. The underlying principle of SEC is that particles of
different sizes will elute (filter) through a stationary phase at different rates. This results in the separation of a
solution of particles based on size. Provided that all the particles are loaded simultaneously or near-
simultaneously, particles of the same size should elute together.
• Each size exclusion column has a range of molecular weights that can be separated. The exclusion limit
defines the molecular weight at the upper end of the column 'working' range and is where molecules are too
large to be trapped in the stationary phase. The lower end of the range is defined by the permeation limit,
which defines the molecular weight of a molecules that is small to penetrate all pores of the stationary phase.
All molecules below this molecular mass are so small that they elute as a single band.

9. ANALYSIS
• For each protein, a volume of the solution needed to elute the protein from the column
called the elution volume(Ve), Ve is proportional to the molecular mass of the protein.
• The elution volume (Ve) decreases roughly linear with the logarithm of the
molecular hydrodynamic volume.
Columns are often calibrated using 4-5 standard samples, and a sample containing a very
large molecule such as thyroglobulin to determine the void volume. The elution volumes of
the standards are divided by the elution volume of the thyroglobulin (Ve/Vo) and plotted
against the log of the standards' molecular weights.

10. III) PROTEIN IDENTIFICATION: OVERVIEW OF PRINCIPLES
• Size determination
Size exclusion chromatography or SDS PAGE
comparison with expected size of protein (known e.g. from reference or cDNA)
• Western Blotting
Binding to specific primary antibody, detected via labelled or enzymatically active
secondary antibody
• Biochemical assays in native gels
Identification of enzymes by their characteristic activity
Identification of metalloproteins by their metal content
• Mass Spectrometry
Fragmentation of the protein, identification of fragment sizes, and subsequent
comparison to a library of known fragmentation patterns
• N-terminal Sequencing (Edman degradation)
Sequential chemical removal of individual amino acids from the N-terminus

11. PROTEIN IDENTIFICATION: MASS
SPECTROMETRY• In a typical MS procedure, a sample, which may be solid, liquid, or gas, is ionized, for
example by bombarding it with electrons. This may cause some of the sample's
molecules to break into charged fragments. These ions are then separated according to
their mass-to-charge ratio, typically by accelerating them and subjecting them to an
electric or magnetic field: ions of the same mass-to-charge ratio will undergo the same
amount of deflection. The ions are detected by a mechanism capable of detecting
charged particles, such as an electron multiplier. Results are displayed as spectra of the
relative abundance of detected ions as a function of the mass-to-charge ratio. The
atoms or molecules in the sample can be identified by correlating known masses to the
identified masses or through a characteristic fragmentation pattern.

12. PRINCIPLE1.protein band is cut of out gel
2.protein is digested into peptides
3. mass spectrometry
4.comparison of the fragment sizes with a database
5. assignment of likely sequences to fragments
6.comparison of the fragment sequences with a database
7. result: list of proteins from the database that have a similar fragmentation pattern

13. PROTEIN CHARACTERISATION: OVERVIEW OF PRINCIPLES
• Size determination
• Charge determination
• Analysis of cofactors
• Analysis of the 3-dimensional Structure
• Activity tests
Protein Characterisation: Analysis of the 3D Structure
Circular Dichroism (CD) Spectroscopy

14. X-RAY CRYSTALLOGRAPHY

15. REFERENCES
• photo of a green gel (Küpper et al., 2003, Funct Plant Biol)
• From: www.columbia.edu/.../c2005/lectures/lec6_09.html
• From: bio-ggs.blogspot.com/2009/11/ggs-live-western...
• From: http://en.wikipedia.org/wiki
• From: elchem.kaist.ac.kr
• Willson and walker (2005) “instrumentation”
• Harper (2008) “fundamental of biochemistry”