Precipitation techniques can be used to recover proteins from liquid and remove unwanted byproducts like nucleic acids. Precipitation is usually induced by neutral salts, organic solvents, changing the pH to the isoelectric point, ionic or non-ionic polymers, metal ions, or heat treatment. The most common precipitating agent is ammonium sulfate, which causes proteins to aggregate and precipitate out of solution by restricting water availability and increasing hydrophobic interactions between proteins.

1. Precipitation
Techniques

2. Precipitation is widely used for recovery of
protein from liquid medium
 Precipitation also be employed for the
removal of certain unwanted byproducts
e.g. nucleic acids, pigments.

3. What is hydration shell of a protein?
It is the surrounding water that is formed around
proteins, also referred to as “protein hydration”,
•This allows homogeneous dispersion in solution and
interactions by hydrogen bonds.
•Also it plays a very important role in protein folding
and function

4. Precipitation is usually induced by
 Neutral Salts
 Organic solvent,
 Isoelectric
 Ionic or non-ionic polymers
Metal ions
Heat treatment

5. Neutral salts
 The precipitation of proteins by salt
 Protein solubility depends on several
factors.
 It is observed that at low concentration
of the salt, solubility of the proteins
usually increases slightly. This is
termed Salting in.

6.  But at high concentrations of salt,
the solubility of the proteins drops
sharply. This is termed Salting
out and the proteins precipitate out.
 The most commonly used salt is
ammonium sulfate, since it is
highly soluble, nontoxic to proteins
and low-priced.

7. Adding the salt to the solution causes
the restriction of the available water molecules for
the proteins, which leads to destruction of the
hydrogen bonds and protein compete
Ammonium sulphate increases hydrophobic
interactions between proteins is stronger than
between the protein and the available water
molecules.

8. This results in protein aggregation and precipitation.
Usually added in the form of solid or saturated
solutions of ammonium sulphate
The precipitation of proteins is dependent on
several factors such as protein concentration, pH and
temperature.

9.  Ammonium sulphate can liberate ammonia
at high pH values and is corrosive to metal
surfaces, e.g. centrifuges.
 The solubility of the salt varies with
temperature, so strict temperature control is
required.

11. II. Organic solvents
If there is a medium decrease in the dielectric
constant with the addition of an organic solvent,
the solubility should decrease also. Here we can
expect precipitation.
Ethanol, acetone and propanol are the
commonly used organic solvents for protein
precipitation
Since proteins are denatured by organic solvents,
the precipitation process has to be carried out
below 0°C.

13. • By changing the pH of the solution, we can
change the charge state of the solute
• pH at which the net charge is neutral is called
the isoelectric point, or pI .
• At a pH below the protein's pI, a protein will
carry a net positive charge; above its pI, it will
carry a net negative charge
• therefore like-charged protein molecules will
exhibit repulsive forces.
III. Isoelectric Precipitation

14. • However, at the pI, the negative and positive
charges cancel, repulsive electrostatic forces
are reduced and the attraction forces
predominate and cause aggregation and
precipitation. Most Proteins pI pH is 4 -6.
• Disadvantages: Irreversible denaturation
occurs when usage of mineral acids ( HCl,
H2SO4)
• Most suitable for removing contaminant
proteins

15. Ionic polymers
The charged polymers such as polyacrylic
acid and polyethylenimine are used.
They form complexes with oppositely
charged protein molecules that causes
charge neutralisation and precipitation

16. Non-ionic polymers
Polyethylene glycol (PEG) is a high
molecular weight non-ionic polymer
that can precipitate proteins.
It reduces the quantity of water
available for protein solvation and
precipitates protein.

17. PEG does not denature proteins,
besides being non-toxic. Other
example dextran
The advantage of using non-ionic
polymers as precipitants is that
they stabilize proteins and may be
used around room temperature.

18. Precipitation by Metal Ions
In this type of precipitation, a metal ion will
bind to a part of the protein.
These ions can be classified into three groups:
Divalent manganese, cobalt, iron, nickel,
copper and zinc bind strongly to carboxylic
acid and to nitrogenous compounds.
Divalent Calcium, Barium and Magnesium
bind to carboxylic acids

19. Divalent silver, mercury and Lead will bind
strongly to sulphydryl groups.
Advantage:
 Have great precipitating power in diluted
solution.
Can be easily removed by cation exchange
resin

20. Precipitation-Heat treatment
• In many cases, unwanted enzyme activities
may be removed by heat treatment.
• Different enzymes have differing susceptibility
to heat denaturation and precipitation.
• Where the required enzyme is relatively heat-
stable allows its easy and rapid purification in
terms of enzymic activity. For such enzymes
heat-treatment is always considered as an
option at an early stage in their purification.

21. • This method successfully applied to the
production of glucose isomerase, where a
short incubation at a relatively high
temperature is used (about 60 - 85C for 10
min).