This document discusses protein purification techniques. It describes that proteins can be extracellular, secreted from cells, or intracellular, requiring cell disruption. Purification techniques include differential centrifugation to separate components by size and mass, differential salt precipitation to separate based on solubility, and various types of chromatography including size exclusion, ion exchange, and affinity chromatography. Electrophoresis can also separate proteins based on size using electrical current. The overall goal is to purify proteins for uses such as commercial products, research, or analysis of structure and function.

1. 25/11/2017

3. Introduction

4. Purpose
include the preparation of commercial
products such as enzymes (e.g. lactase),
nutritional proteins (e.g. soy protein
isolate),
certain biopharmaceuticals (e.g. insulin).
research or analytical purposes,
 including identification,
 quantification,
and studies of the protein's structure,
 post-translational modifications and
function.

5. Separation:-
Extracellular:-
•No need for cell disruption
•Secreted soluble proteins can be collected
in the cell supernatant after centrifugation
•Membrane-bound proteins might be
released from the cell simply using
detergents

7. Need cell disruption
•Detergents lysis
•Enzymatic lysis
•Osmotic lysis
•Ultrasonication
Intracellular

8. Purification:-
oDifferential centrifugation:-
Force Time sediment
1000g 5min Eu. cell
4,000g 10min Chl,cell debris,
nuclei
15,000 20min Bacteria, mito
30,000 30min Small
organelle
200,000 1hr Small vesicle
100,000 3-10hrs Ribosome
200,000 10-24hrs Membrane
sheets

9. Differential salt precipitation

10. oDifferent chromatography
Column Chromatography :-
•Most common and best approach to purifying
larger amounts of proteins
•Achieves highest level of purity and largest
amount
•Requires low effort
•Lowest likelihood to damage the protein
product
•Standard method for pharmaceutical industry

11. Size-Exclusion (or molecular
exclusion) Chromatography
•Molecules are separated
according to differences in their
size as they pass through a
hydrophilic polymer
•Polymer beads composed of
cross-linked dextran (dextrose)
which is highly and uniformly
porous (like Swiss cheese)
•Large proteins come out first
(can’t fit in pores), small proteins
come out last (get stuck in the
pores)

12. •Principle is to separate
on basis of charge
“adsorption”
•Highest resolving power
•Highest loading capacity
•Widespread applicability
•Most frequent
chromatographic
technique for protein
purification
•Ion Exchange chromatography

13. Adsorptive separation in which
the molecule to be purified
specifically and reversibly
binds (adsorbs) to a
complementary binding
substance (a ligand)
immobilized on an insoluble
support (a matrix or resin)
•Purification is 1000X or better
from a single step (highest of
all methods)
•Preferred method if possible
•Affinity chromatography

14. Polyacrylamide Gel Electrophoresis
oElectrophoresis separates proteins based on their size using electrical current

16. protein are usually soluble in water solution
because they have hydrophilic amino acids
on their surface that attract water molecules
and interact with them.
Conclusion

17. Reference:-
•Bioanalytical techniques, M. L.
Srivastava(2008), Narosa Publication House,
ISBN 978-81-7319-852-6
•Principles of Biochemistry, David L. Nelson
And Michael M. Cox (2008), W H FREEMAN
And Company, ISBN-10: 0-7167-7108-X
•Molecular Cell Biology, LODISH And et.al.
(2016), W H FREEMAN And Company,
ISBN10:1-4641-8339-2