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Theory module: 09
PRECIPITATION OF PROTEINS AND NUCLEIC ACIDS
Introduction:
Analyses of proteins and nucleic acids play an important role in modern biology.
Thousands of proteins and different nucleic acids direct the whole cellular process. To
study and analyses the number of proteins and nucleic acid at different stage of cellular
development it is necessary to extract it quantitatively. Precipitation is widely used for
the recovery of biomolecules. Precipitation is usually induced by addition of a salt or an
organic solvent or by changing the pH to alter the nature of the solution.
Precipitation of proteins
a. Precipitation of proteins by salts
The most common type of precipitation for proteins is salt induced precipitation.
Different types of salts such as ammonium sulphate and sodium sulphate are widely
used to precipitate out proteins . Ammonium sulphate is the most widely used salt for
the precipitation of proteins as it is highly soluble, inexpensive, available in highest
purity level, does not change the protein solution to extreme pHs and in most of case it
does not denature proteins.
Ammonium sulphate can be used for precipitation of total proteins at ~90% saturation
or for differential precipitation level of proteins using different saturation of salts. Up to
20% saturation, ammonium sulphate precipitate particulate materials, and
preaggregated and very high molecular weight proteins and at 90% saturation it
precipitates almost all proteins.
Principle of protein precipitation by salts
Proteins have polar amino acids such as glycine, serine etc. Usually in native proteins
hydrophilic amino acids are on the surface of proteins whereas hydrophobic amino
acids are buried. Attractive interactions between the nearby oppositely charged groups
are ion pairs or salt bridges. Analysis has revealed that in folded proteins, 4 attractive
ion pairs and 1 repulsive ion pair are present per100 amino acids. Water as powerful
solvent, interacts with these surface amino acids and keep them in solution.
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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. But
at high concentrations of salt, the solubility of the proteins drops sharply. This is termed
Salting out and the proteins precipitate out. During ammonium sulphate precipitation
the salt has to be added in small amount under constant stirring to avoid accumulation
of high concentration of salts. When large amount of salt is added to an aqueous
solution of proteins the salt requires more amount of water for its dissolution. This
leads to competition for water molecule on the proteins. Completely ionized salts have
more affinity for water molecules then protein hence addition of salts takes up water
molecule from the protein. Therefore the ionic interactions between water molecules
and protein are reduced and as result hydrophobic interactions dominate. The
hydrophobic amino acid patches present in all the proteins attract each other and forms
aggregates. These aggregates are nothing but the proteins in the form of precipitates.
In salt precipitation, the anions appear to be more significant. Temperature, pH and the
most important the protein concentration affect ammonium sulphate precipitation of
proteins to large extent. Higher ammonium sulphate is required for precipitating highly
soluble proteins.
b. Precipitation of proteins by organic solvents
Another method is the addition of solvent. Water miscible organic solvents like ethanol,
methanol and acetone are also good protein precipitations. The principle of solvent
precipitation is different from salt. 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.
Three important criteria which should be considered when using organic solvent as a
precipitation is as follows
1.
2.
3.
4.
Solvent should be completely miscible with water
Solvent should be available in highest purity
Solvent should not react with proteins
Solvent should have a good precipitating effects
The most commonly used solvents is acetone and ethanol but acetone is better protein
precipitant than ethanol. Precipitation using organic solvent is done at 0 oC or subzero
temperatures in order to minimize the denaturation of proteins. In this method solvent
is cooled overnight and added slowly with constant stirring to ice-cold solution of
proteins in cold condition. The precipitates are collected by centrifugation at 10,000
rpm for 20 mins at 4oC. Advantage of using solvent precipitation is that it can be easily
removed by evaporation
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Principle of protein precipitation by organic solvent
Water keeps protein molecules in solution by minimizing the electrostatic attractions
between them. Water molecules reduce the strength of electrostatic attraction by a factor
of 80, the dielectric constant of water at 0oC. The principle effect of solvents is to reduce
the dielectric constant of water during precipitation. Thus solvent minimizes the
solvating capacity of water molecules by reducing their dielectric constant. Hence
when the concentration of solvent increases it brings about bulk displacement of water
molecule. This allows strong hydrophobic interactions between proteins and the
hydrophobic patches of different proteins interact and aggregate.
c. Precipitation of proteins by organic polymer
Water soluble high molecular weight organic polymers are also used to precipitate
proteins. PEG (poly ethylene glycol) is widely used for the precipitation of proteins as
well as for nucleic acids. PEG are linear polymers of ethylene glycol {HOCH2-(CH2CH2-O-)n-CH2OH}
PEG is anionic and water soluble so it does not react with proteins and nucleic acids
hence produce minimal denaturation effect on macromolecules. Lower concentration of
PEG precipitates higher molecular weight proteins and nucleic acids and vice versa.
PEG with different MW is available but PEG with MW more than 4000 are very
effective for precipitation. The most important factors for precipitation are pH and salt
concentrations. Much lower concentration of PEG is enough when proteins are at their
isoelectric points. Salts concentration also influence precipitation by PEG.
Principle of precipitation by PEG
The principle of precipitation of PEG is not well understood. It is assumed that PEG
possible brings about bulk displacement of water molecules like organic solvents and
immobilizes them. It may also involve in reducing the water activity like the organic
solvents. They have stabilizing effects on biomolecules which is possibly because of
polyhydroxyl groups. The polymerized molecule favors the hydrophobic interactions
among proteins and nucleic acids which results in their precipitation.
d. Isoelectric precipitation of proteins
Solubility of proteins is the results of polar interactions with water molecules and to
some extent the repulsive electrostatic forces between the protein molecules themselves,
if the repulsive electrostatic forces between the protein molecules are insufficient then
the protein molecules together and forms the precipitate.
Another method is precipitation by changing the pH of the protein solution. This effect
is due to the different functional groups on a protein. There will be some pH, known as
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the isoeletric point where the net charge on the protein is zero. This is different for
different proteins. When the pH of the medium is less than the isoelectric point of a
protein, the protein molecules will carry a net positive charge. When the pH of the
medium is more the isoelectric point of a protein, the protein molecules will carry a net
negative charge. As the like charges repel each other, the protein molecules stay in the
solution at pH above and below the IEP. But when the pH of the medium is equal to
IEP, all the charges on the proteins are neutralized hence in absence of any repulsive
force, the protein molecules get together and precipitate out.
Precipitation of nucleic acids
Nucleic acids are generally precipitated by ethanol, isopropanol or PEG. The most
commonly used solvent for nucleic acid is ethanol. It is simple, rapid and sensitive as it
can precipitates nanogram quantity of DNA and RNA in few minutes. Residual ethanol
can be easily removed by air drying or vacuum drying the pellets for few minutes.
DNA concentration as low as nanogram can also be easily precipitated under standard
conditions. For effective precipitation of nucleic acids monovalent cations like
ammonium acetate or lithium chloride or sodium chloride or sodium acetate is added
before the addition of cold ethanol. Buffers containing high concentration of EDTA
should be avoided during ethanol precipitation as it gets precipitates along with nucleic
acids. RNA is also efficiently precipitated with the above mentioned cations with 2.53.0 volumes of cold ethanol.
a. Precipitation using isoproponal
Large volumes of DNA samples can be efficiently precipitated using one volumes of
isoproponal at 0oC and incubating the solution for ~30 mins along with the monovalent
cations.
b. Precipitation using PEG
PEG a water soluble organic polymer is used for the precipitation of DNA. High
molecular weight DNA can be precipitated with low concentration of polymer and vice
versa. PEG enhances hydrophobic interactions among DNA molecules and brings them
closer in the form of precipitates. This is evident form its use in blunt end ligation
reactions and the phenomenon is known as molecular crowding.
c. Precipitation using polyethyleneimine (PEI)
PEI is a highly branched synthetic cationic polymer, effective in nucleic acid
precipitation especially from cell extracts. PEI at 0.3% precipitates 90% RNA and 96%
DNA at pH 7. (PEI is a carcinogenic because of unreacted monomers).
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d. Precipitation using cetyl trimethyl ammonium bromide (CTAB)
CTAB is a cationic detergent especially used for extraction of DNA from DNA
fragments. CTAB binds DNA strongly and forms an insoluble complex. CTAB has a
strong affinity for DNA as it carries positive charge and DNA carries negative charge
and hence precipitates out DNA. In this method of precipitation salt is concentration is
very important. When the concentration of salt is above 1M (NaCl) no precipitates is
formed but when it is less than 0.2M all nucleic acid is precipitated. If the concentration
of salt is maintained between 0.3 to 0.4 then small DNA fragments and oligo primers
are not precipitated. Hence CTAB provides a suitable way of extracting different size of
DNAs. CTAB also solubilizes membranes and therefore it is used in the extraction
buffer used for lysis of cell.
e. Precipitation using trichloroacetic acid (TCA)
TCA precipitates nucleic acid and proteins efficiently at a concentration of 5% to 10% at
0oC. Generally 20%-50% stock solution of TCA is mixed with equal volume of the
sample which gives final concentration to 5%-10% and incubated at 0oC for 30mins to
gives complete precipitation.
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