Biochemistry Lab Report 1
Spectrophotometry, Quantification of Protein Concentration, Chromatography, Gel
Electrophoresis, Gel Electrophoresis of Proteins
Abstract
Over the course of three lab periods, many experimen
...
Biochemistry Lab Report 1
Spectrophotometry, Quantification of Protein Concentration, Chromatography, Gel
Electrophoresis, Gel Electrophoresis of Proteins
Abstract
Over the course of three lab periods, many experiments were performed in order to set up
a stable base for this class of Biochemistry lab. To do this, we made many measurements to
create many standard graphs so that we have them in our arsenal for future experiments. In order
to perform detailed studies on the structure and function of specific biomolecules, we must first
be able to purify the biomolecule of interest. Some methods of purification include Affinity
Chromatography and Size Exclusion Chromatography. After, we must perform qualitative and
quantitative tests on these compounds of interest to understand how much of it is present and
what are some of its features. A range of methods and devices can be utilized to preform these
tests. Quantitative tests are especially important because they tell us how well we have purified
the compound of interest. A great device used to perform these tests is the spectrophotometer. We
can also use a gel electrophoresis to determine whether multiple proteins are present after our
attempts to purify proteins.
Introduction
Throughout many lab periods, multiple new techniques were used in order to perform
both qualitative and quantitative analyses of a few different compounds of interest. These
important laboratory techniques enable us to attain valuable information about our desired
compound such as absorbance, concentration, pH, and activity. One of the most important
techniques used was the purification of a specific compound.
The first technique that was used was Spectrophotometry. The purpose of this technique
is to determine the amount of light that is absorbed by a specific compound at a specific
wavelength or over a range of wavelengths. In order to properly measure the amount of light
absorbed by a specific compound, a “blank” is usually tested first. The blank contains everything
that the cuvette with the compound has (buffer, etc.) except it lacks the compound. By lacking
the compound, it can account for the absorption of light that takes place due to other compoundsBiochemistry Lab Report 1 2
in the cuvette, thus providing us the precise absorption of the compound of interest within the
cuvette. There are different types of spectrophotometers but the one that we used is known as
“Spectronic-20”. This type of spectrophotometer can only test in a range of 340 nm – 650 nm
wavelengths. Within compounds, there exist many different bonds and electrons that absorb light
at specific wavelengths. A very specific amount of light of the right energy will excite the
compound. The light that the spectrophotometer shoots out is known as the “incident light” (Io).
This light is then split into “transmitted light” (I), which is read by the spectrophotometer and the
absorbed light.
These important numbers are part of an equation known as “Beer’s Law”. Qualitatively,
this law simply means that the amount of light absorbed by the compound is proportional to the
concentration of a colored compound. Quantitatively however, this law is written as A = Ecl.
Where E is the absorption coefficient, c is the concentration, l is the path length, and A is the
absorption. In our lab, our compound of interest was p-nitrophenol (PNP). The first thing we did,
Experiment 2.1, was test a known concentration of basic and acidic forms of PNP at different
wavelengths on the spectrophotometer in order to find the where its absorption was highest, also
known as maximum absorption. By finding this critical wavelength, we were able to use it in
Experiment 2.2 as a constant when testing PNP at different pHs. After testing the absorption at
different pHs, we are able to make a pH vs. Absorption graph and to find the pKa of PNP. The
pKa of a compound is the pH where there are equal concentrations of basic and acidic forms of
the compound. The maximum absorption was also used in Experiment 2.3 to test the absorption
of PNP at different concentrations (but constant pH) in order to form a concentration vs.
Absorption graph. Using this graph, we could find the concentration of an unknown amount of
PNP from its absorption. In experiment 2.4, we will use the graph from experiment 2.2 (pH vs.
Absorbance) in order to find the pH of a solution that PNP is mixed with.
The whole point of quantifying the concentration of proteins is to determine how well we
purified a protein. If the protein we are looking for is an enzyme, we could also test its activity to
see how concentrated it is. One method of protein quantification is known as the Bradford
Method. This was utilized in experiment 3.1, in order to determine the concentration of an
unknown amount of Bovine Serum Albumen (BSA). A dye known as “Coomasie Brilliant Blue”
is used, and although it normally has absorption of 465 nm, when it is mixed with proteins, it
forms non-covalent bonds that bump its absorption up to 595 nm. This phenomenon is known as3
“Red Shift.” Some of these non-covalent bonds include Van Der Waals and electrostatic
interactions. In order to find the concentration of the unknown BSA, the absorbance must be
taken at 595 nm and compared to the standard curve of known concentrations of BSA vs.
Absorbance.
An important method in purifying proteins is Chromatography. Using this method, you
can separate molecules based on the molecular weight, their charge, or even their affinities to
other molecules. There are two phases in chromatography: a stationary phase and a mobile
phase. In the stationary phase, the column is filled with a gel or bead-like mixture specific to the
type of chromatography you want to perform or molecule you want to purify. In the mobile
phase, a solvent passes through the column
[Show More]
 (1).png)
.png)
.png)
