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ABSTRACT
In this experiment, qualitative analysis through paper chromatography was done and Bradford
total protein assay for quantitative analysis. Paper Chromatography is a type of partition chromatography
that uses a specialized paper made up of polymeric carbohydrate cellulose (filter paper) as a support
medium for the stationary phase. It is generally used for the separation of water-soluble organic and
inorganic compounds or highly polar compounds such as amino acids and sugars. This method is useful
for separating complex mixtures of compounds having similar polarity, like amino acids. In this experiment
10 amino acid standards were used to compare and determine as to which amino acid is present in the
protein casein obtained from milk. The Rf values were computed and it was seen that the enzymatic
hydrolysate obtained from casein has the same Rf value with proline which means that casein contains is
proline. The Micro-Bradford Protein Assay, on the other hand, is a rapid and accurate method for the
estimation of protein concentration. A total of 6 test tubes were prepared with different volumes of the
standard and water, and the other test tube contained the unknown sample, respectively. In a microplate
(96-well), each cuvettes contained the BSA standard and were filled with a right amount of each test tube
prepared and was immediately subjected to the UV/Vis Spectrophotometer. The absorbance of each
sample was determined and plotted into a linear graph. The concentration of the sample was also
computed using the linear regression method and resulted to 0.7843 mg/mL.
INTRODUCTION
For Paper Chromatography, a 12x15cm reagent were added to each cuvettes in the
of filter paper was prepared and from the bottom microwell plate (Figure 1), enough volume of the
of the longer edge, a 1.5 cm margin was made samples from the test tubes made were then
to serve as the origin of the amino acid transferred to the cuvettes with Bradford reagent
standards and the enzyme hydrolysate to be and was mixed well. The first cuvette served as
dropped. On the line made, 11 equidistant points the blank while the rest of the cuvettes
were marked for the spotting of the respective contained the samples. The albumin sample
samples to be used. Using a capillary tube, the which was made by mixing a 75L of Bradford
amino acids standards were dropped 5 times reagent and 100L of the BSA standard, was
and the enzymatic hydrolysate 10 times. After all also pipetted into the cuvettes three time to
the samples were dropped and let dried, the obtain an accurate result. The microplate was
chromatogram was stapled properly together then placed inside the spectrophotometer and
forming a cylinder and was placed inside a was read at the absorbance 595nm. The
1000mL beaker with the solvent butanol:acetic albumin standard curve was then constructed by
acid:water in the ratio of 4:1:5 just below the plotting the absorbance at 595nm against
origin and was covered with a watch glass until concentration and the concentration of the
the solvent front was approximately 0.5 cm from protein sample was determined.
Trial 2 0.8140 0.4470 0.6265 the absorbance. The Beer-Lambert law relates
the attenuation of light to the properties of the
Trial 3 0.8300 0.4630 0.6683
material through which the light is traveling. This
Average 0.465 0.6474 page takes a brief look at the Beer-Lambert Law
and explains the use of the terms absorbance
In determining the total protein
and molar absorptivity relating to UV-visible
concentration of the sample, the linear
absorption spectrometry.[12]
regression (y=mx+b) was used to compute for
the slope and the y-intercept then the
concentration which is the variable x. The slope
having a value of 0.3823, 0.2075 for the y-
intercept and 0.7843 for the concentration of the CONCLUSION
protein sample. However, the total protein
From the experiment performed, based
concentration can also just be manually
on paper chromatography it can be concluded
inspected and in this experiment it gave a
that the protein casein isolated from a non-fat
concentration of 0.6474mg/mL.
skimmed milk contains the amino acid proline
because proline has the same Rf values with the
enzymatic hydrolysate obtained from casein.
While in the Micro-Bradford Total Protein Assay,
it can be concluded that the protein
concentration of the unknown sample is
0,7843mg/mL when computed and 0.6474 when
manually inspected.
REFERENCES
[1]
Kumar, Paul. (n.d.). Biochemistry:
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