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New Microsoft Word Document

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hibakhalidllcc
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1. Which fraction contains the most proteins?

Protein signals are expected to be most pronounced in the fractions with


higher salt concentrations because it disturbs the binding of the protein to
the column. As shown in figure1, fractions 3 and 4 have more pronounced
darker colour indicating the possibility of containing the highest protein
concentration which can be confirmed by the red660 assay. Cytochrome c
is more positively charged than haemoglobin so it would bound strongly to
the cation exchange resin and would require high salt concentration to
elute it. Whereas haemoglobin would be eluted earlier.

2. Describe briefly how proteins are immobilized on an ion-exchange


column.

The immobilization of proteins occurs as a result of their charge at a


specific pH level. In this experiment, a cation-exchange column is utilized,
which captures proteins with positive charges through the substitution of
counter-ions within the column's matrix. This capture process is
dependent on ionic interactions between the positively charged regions of
the proteins and the negatively charged components of the column
matrix.

3. Describe the elution process for immobilized proteins.

The process of elution is accomplished through a gradual increase in the


salt concentration of the buffer solution. As the salt concentration rises, its
ions compete with proteins for binding to the charged sites on the column.
Proteins with weaker interactions are eluted first, while those with
stronger binding affinities require higher salt concentrations to be
displaced and eluted from the column.

4. Predict the elution profile if the pH of the Elution Buffer is altered.

The alteration of pH leads to a change in protein charge via protonation


which means adding a proton or deprotonation removing a proton
processes. For example, at a lower pH, proteins could become relatively
positively charged and this could Favor them in binding to the cation-
exchange column. As a result, this could postpone the time when proteins
are eluted from the column. On the other hand, when the protein is in a
higher pH environment, the proteins may have lost positive charge or
acquired negative charges which will lead to less binding and earlier
elution of proteins.

5. How is the operating pH selected for ion-exchange chromatography?


The pH of the wash is chosen according to the isoelectric points (pI) of the
proteins of interest. This further means that, at the appropriate pH, the
targeted protein should possess a given charge (positive when cation
exchange is applied, and negative when anion exchange is done) so as to
make it able to attach onto the column while the impurities are not
adsorbed or are washed out under different specifications.

Ion exchange chromatography is a pivotal technique in proteomics,


enabling the separation and purification of proteins based on their charge
properties. It relies on the principle that biomolecules with opposite
charges adhere to ionic groups immobilized on a chromatographic column.
Proteins, which have variable charges depending on their amino acid
composition and pH of the environment, can be selectively bound and
eluted by adjusting these conditions. This method is crucial for isolating
individual proteins from complex mixtures, thereby facilitating the study
of their unique structures and functions. Of its application. Ion exchange
chromatography is employed in a variety of industries, including the
pharmaceutical, petrochemical, and agricultural sectors, in addition to
analytical chemistry and biological chemistry for the separation of cations
and anions. There are two types of exchangers, cationic exchangers, that
have a negatively charged resins or column that would attract the
positively charged cations. Anionic exchangers have negatively charged
resins or columns that would attract negatively charged anions. In this lab,
the focus is on the separation of Cytochrome C and hemoglobin using a
cation exchange column to explore the technique's utility in protein
purification.

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