1. Protein purification involves isolating one or a few proteins from a complex mixture through a series of extraction, precipitation, ultracentrifugation, and chromatographic steps. 2. Maintaining the proper buffering environment is essential to prevent changes in pH that could denature proteins. Buffers should be selected so their pH is within 1 unit of the protein's pKa. 3. When proteins are purified, they should be stored with additives at the appropriate pH and temperature depending on the protein's stability requirements for downstream applications. Storage conditions need to be optimized for each protein.

1. B Y H A L A V A T H R A M E S H
1 6 - M C H - 0 0 1
D E P A R T M E N T O F C H E M I S T R Y
L O Y O L A C O L L E G E – C H E N N A I
U N I V E R S I T Y O F M A D R A S
Protein Purification

2. Introduction
 Protein purification is a series of processes intended to isolate one or a few proteins
from a complex mixture.
 The purification process may separate the protein and non-protein part of the mixture
and finally separate the desired protein from all other proteins.
 The protein isolation is depends on protein size, physico-chemical properties binding
affinity and biological activity.
 Protein purification mainly four steps
 1.Extraction
 2.Precipitation and differential solubilisation (salting out)
 3. Utracentrification
 4.Chromatographic methods.

3. Buffering component: The solution conditions of a protein at each step of the purification scheme are
essential in maintaining protein stability and function. Proteins should be kept in a well-buffered
environment to prevent sudden changes in pH that could irreversibly affect their folding, solubility, and
function. A buffer is a solution containing a conjugate acid/base pair. The pH range of a buffer is based on
its pKa, defined as the pH at which 50% of the molecules are in their acidic form, and 50% are in their
basic form. A general rule regarding buffers is that the pH of the buffer solution should be within 1.0 pH
unit of the pKa to provide appropriate buffering capacity. This ensures that there is a sufficient amount of
the molecule in both its acidic and basic forms to neutralize the solution in case of H+ or OH- influx. Thus,
buffers prevent pH changes that could negatively affect protein stability. Typically, these buffers are used
at concentrations above 25mM to ensure adequate buffering capacity. List of some biological buffers

5. Sonication : Sonication involves inserting a tip into a test tube or eppendorf and sending a
high intensity sound wave throughout the sample to disrupt cell walls and shatter tissues.
Sonication is easy, and it can be applied to samples of almost any size, but it should be used
with care, since this method may cause proteins to overheat and denature (breakdown). We
recommend using multiple, short sonic bursts as opposed to one long burst. All sonications
should be performed on ice and the samples allowed to rest and chill between bursts.
Chelating: Chelating is a type of bonding of ions and molecules to metal ions. It
involves the formation or presence of two or more separate co-ordinate bonds
between a poly dentate ligand and a single central atoms. Chelating agents are
chemical compounds that react with metal ions to form a stable, water –soluble
complex. Chelating agents have a ring –like center which forms at least two bonds
with the metal ion allowing it to be excreted.

6. Additives commonly used in protein purification buffers to increase the stability of
proteins.

7. Affinity chromatography:
Affinity chromatography relies on the specific and reversible
binding of a protein to a matrix-bound ligand. The ligand can bind directly to either
the protein of interest or a tag that is covalently attached to the protein. Affinity
chromatography is often the most robust purification procedure and is typically used
in the early stages of the purification scheme. Depending on the downstream
application, affinity purification might be the only chromatographic step required to
achieve adequate purity.
The stationary phase for affinity chromatography is
made of an inert matrix covalently attached to a ligand that specifically binds to a
protein or group of proteins. The inert matrix is typically composed of cross-linked
agarose or polyacrylamide. Proteins can be purified by affinity chromatography in a
selective or non-selective manner. In selective affinity chromatography, a ligand
specific for a protein or a covalently attached tag is used. In non-selective affinity
chromatography such as Protein A, G, L for immunoglobulin, or heparin for DNA-
binding proteins, or lectin for glycoproteins, the ligand binds to a group of proteins
with similar binding capabilities.

8. Dielectric Point: The maximum electric field that the material can withstand under ideal
conditions without breaking down.
Autoxidation: Spontaneous oxidation of a substance at ambient temperature in the
presences of oxygen.

12. Summary:
1. A buffer is a solution containing a conjugate acid/base pair.
2. When proteins are deemed pure enough for use in experimental studies, they
should be stored appropriately. The selection of a final storage buffer is just as
important as the selection of buffers used during the purification scheme and
should depend on the stability of the protein and conditions required for the
downstream application of the purified protein. Often, size exclusion
chromatography is selected as a final step in the purification scheme, as the
storage buffer can be used in this chromatographic step to exchange the buffer
effectively. The pure fractions can be pooled for immediate storage.
Alternatively, the final pooled fractions can be dialyzed into the selected buffer
before storage.
3. Protein storage conditions depend on the protein of interest and should be
optimized, so the protein maintains structural and functional stability over long
periods of storage. Additives are often included in the storage buffer to enhance
the lifetime of purified proteins under storage conditions, and trial and error are
often required to determine optimum conditions, as every protein behaves
differently.

13. Why is EDTA used as a chelating agent ?
Ans: EDTA is a versatile chelating agent. It can form four or six bonds with a metal ion,
and it forms chelates with both transition metal ions and main group ions. EDTA deactives
these enzyme by removing the metal ions from them and forming stable chelates with them.
Chelating agents
1. EDTA
2. Oxalic acid
3. NH3
4. Thiosulphate
PH= 7 below……..Acidic
PH=7 Neutral
PH= 7 greater ……Basic
References: https://www.labome.com/method/Protein-Purification.html