Proteins Are One of The Four Macromolecule Building Blocks of Life. The Other Three Are

INTRODUCTION
Proteins are one of the four macromolecule building blocks of life. The other three are
carbohydrates, lipids, and nucleic acids. Proteins are long strings of amino acids that fold together into
hierarchal structures in order to perform specialized functions within the cells and tissues of all living
organisms. These higher structures are imperative to the proper function of the protein within its
biochemical pathway. The tertiary structure creates the chemical and morphological landscape that
imparts the biorecognition abilities to ligand, receptors, antibodies, and all the other workhorse proteins in
the organism. Estimation of protein concentration is necessary in protein purification, electrophoresis, cell
biology, molecular biology and other research applications.
Protein assays are one of the most widely used methods in life science research. Although there
are a wide variety of protein assays available, none of the assays can be used without first considering
their suitability for the application. Each assay has its own advantages and limitations and often it is
necessary to obtain more than one type of protein assay for research applications. A specific solution
would probably come up with different findings if measured using different methods for a reason that they
are based on different principles. An absolute method does not exist; everyone has some advantages
and disadvantages. Also, the nature of the proteins present in the sample, the purity of the extracts, on
the required sensibility and accuracy, and on the desired speed is the basis for the choice of an adequate
method (Boyer, 1986). Some of the widely known utilized method for the protein content analysis are the
assays of Biuret, Bradford, Kjendahl, Lowry, Smith, and Warburg-Christian. However, most of the authors
propose the spectrophotometric assay of Bradford (1976) because of its multiple advantages if compared
with other methods (Snyder and Desborough,1978; Berges et al., 1993; and see Bio-Rad bulletin
1069EG, 1979).
The Bradford method is based on the absorbance shift observed in acidic solution of dye
Coomassie Brilliant Blue G-250. When added to a solution of protein, the dye binds to the protein resulting
in a change of color. The dye has been assumed to bind to protein via electrostatic attraction of dye’s
sulfonic groups to the protein. The bound points are primarily arginine residues, but the dye also binds to a
lesser degree to histidine, lysine, tyrosine, tryptophan and phenylalanine (Compton and Jones, 1985). The
peak absorbance of the acidic dye solution changes from 465 to 595 nm when binding to protein occurs.
Therefore, measuring absorbance of the protein-dye complex at 595 nm allows an accurate quantification
of the protein content of a sample.
Advantages of Bradford method are: (i) the use of a single reactive, (ii) the rapidity of the reaction
(just 5 min.), (iii) a high stability of the protein-dye complex, (iv) a high reproducibility, and (v) the occurrence
of minimal interferences. This rapid and economical process has various applications in experimental
sciences. Further, Bradford method is well-suited for repetitive determination of protein concentrations (e.g.,
in physiological, immunological, cytological, clinical, and food quality routine analysis), this assay is often
linked to other techniques. This also serves as a convenient means of monitoring column chromatography
effluents and, due to the relative insensitivity to interferences, it is a valuable method for analyzing fractions
from affinity, ion exchange, gel and adsorption chromatography. This protein assay offers a valuable
method of examining sample loads for gel electrophoresis. The method also abridges the evaluation of
protein concentration during enzyme purification of different tissues. Moreover, the Bradford assay is
adaptable to semiautomated methods of protein quantification.
ANSWERS TO RESEARCH QUESTIONS
A. What limitations of image analysis have you observed? What is an appropriate plan to
resolve this?
In image analysis, it is a set of techniques that allows somebody to obtain quantitative
results coming from morphological preparations. It is a powerful tool capable of providing
meaningful data but can provide erroneous results when not used properly and correctly. The
techniques used in image analysis is highly dependent on computer processing thus many errors
are introduced inadvertently with profound results. Another limitation is that it requires
assumptions to be made and only provides measurements of relative changes to the objects of
interest. One way of resolving this is to carefully prepare assays and have an analytical thinking
to have accurate and efficient results.
B. What is the principle behind Bradford Assay for determining the concentration of protein
in your samples? How do you minimize errors caused by interference of other compounds
in a complex chemical matrix using Bradford Assay?
Bradford Assay is a quick and accurate spectroscopic analytical procedure used to
measure the concentration of protein in a solution. The reaction is dependent on the amino acid
composition of the measured proteins. The principle behind is based on the proportional binding
of the dye Coomassie to proteins. The more protein present, the more Coomassie binds.
Furthermore, the assay is colorimetric; as the protein concentration increases, the color of the
test sample becomes darker. The protein concentration of a test sample is determined by
comparison to that of a series of protein standards known to reproducibly exhibit a linear
absorbance profile in this assay. Although different protein standards can be used, we have
chosen the most widely used protein as our standard - Bovine Serum Albumin (BSA). To lessen
errors and false positive results, one must prevent the stock solutions to be contaminated of other
compounds that can also react with Coomassie blue.

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INTRODUCTION

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