National University

College of Allied Health

Term 1, AY 2022-2023

LABORATORY JOURNAL No. 1

Protein Purification

Name: Section: Matel, Danielle Anne C. Student ID No.: 2022-109092

Instructor: Ms. Rianne Chenee S. Rico

I. Materials Used (please attach actual photo/screenshot from the video of all materials utilized)

II. Post-lab questions:

1. Discuss Proteins (2pts)

Compared to other kinds of macromolecules, proteins are among the most prevalent organic molecules in
biological systems and have a much wider range of structural and functional variations. A single cell has
hundreds of proteins, each with a unique function. Although proteins' shapes and functions vary greatly
from one another, all proteins are made up of one or more chains of amino acids. In this post, we'll delve
into further detail on the parts, arrangements, and purposes of proteins.

2. Discuss the relationship between amino acids and proteins (3pts)

Proteins are made up of monomeric amino acids, which are known as protein building blocks. As tiny
chemical molecules with an alpha carbon atom connected to an amino group, a carboxyl group, a
hydrogen atom, and a variable side chain, amino acids are regarded as such. A lengthy chain of amino
acids is formed inside the protein by the peptide bonds connecting various amino acids. The main
structure of a protein is the orderly arrangement of amino acids within it.

3. Discuss the environmental factors that influence solubility of proteins(5pts)

The concentration of proteins in a saturated solution that is in equilibrium with a solid phase, either
crystalline or amorphous, under specific conditions, is known as protein solubility. Solubility can be
influenced by a wide range of internal and environmental factors. The presence of different solvent
additives, temperature, pH, ionic strength, and ionic strength are all extrinsic factors that affect protein
solubility. Although adjusting the solution conditions can sometimes be acceptable or sufficient to
increase protein solubility to the desired level, hanging these external characteristics can improve protein
solubility. For structural biologists, the pharmaceutical industry, and all other scientists who work with
proteins in solution, protein solubility is essential. Protein samples with incredibly high concentrations are
routinely needed for pharmacological applications and structural analysis.

4. Enumerate the techniques for protein extraction (5pts)

 hydrophobic interaction column chromatography, size exclusion chromatography, ion exchange column
chromatography, and affinity chromatography.
 5. Discuss the protocol for purifying a protein of interest (5pts)

The process starts with the preparation of the sample, which consists of cell harvesting, cell disruption (in
the case our target protein is intracellular), and clarification. Cell harvesting involves separating the cells
from the culture medium, usually by centrifugation or filtration. As for the cell disruption, it can be
achieved through different methodologies that will be chosen depending on the host. Not all cells
demonstrate the same resistance to lysis, and this should be taken into account because more rigorous
protocols are needed for the more resistant cells (ex: bacterial cells, due to their cell walls). The most
widely used techniques are chemical methods (such as enzymatic methods or detergents) and physical
methods such as sonication. Every technique has benefits and drawbacks, and no approach can ever yield
a sample that is 100 percent pure. For this reason, a target protein is considered to be produced by many
protein purification procedures as a "enriched population" as opposed to a pure population. As a result,
we frequently combine different techniques, like sonication followed by a detergent treatment. Naturally,
cell disruption won't be necessary when preparing a sample of secreted proteins, eliminating a step.
Clarification is the final step in the preparation process because the protein that needs to be purified will
likely mix with other substances like membrane fragments, organelles, cellular waste, or insoluble
proteins. This is accomplished through filtration or centrifugation, which separates the liquid fraction with
dissolved soluble molecules from the heavier intact cells using centrifugal force. In order to do the
separation itself, we need to get a clean medium free of particles. We try to lower the working volume
during this process so that we can deal with volumes that are easier to handle. But in order to boost
production, the volume must be increased, as is frequently the case in industrial settings. Large volumes
of proteins and cells can behave differently than small volumes, making the scaling-up process
challenging. This is especially true if the capture step, which comes after the biomagnetic separation step,
is used.

6. Discuss the principle, procedure and application of column chromatography (5pts)

Column chromatography is a helpful technique in which the compounds to be isolated are introduced onto
the highest point of a column loaded with an adsorbent, pass through the column at varying rates based on
the affinities of each substance for the adsorbent and the solvent or mixture, and are commonly gathered
in solution at different periods as they move through the column. Silica gel and alumina are the two most
prevalent examples of stationary phases for column chromatography, whilst organic solvents are thought
to be the most prevalent mobile phases. The primary principle involved in column chromatography is the
adsorption of the solution's solutes with the help of a stationary phase, which then separates the mixture
into independent components. When the mobile phase and the mixture that has to be isolated are lowered
from the top of the column, the movement of the mixture's different components occurs at varying rates.
Comparatively speaking, the components with lower adsorption and affinity to the stationary phase leave
the mixture more quickly than those with higher adsorption and affinity. The elements that move quickly
are removed first, while the elements that move slowly are removed last.