Mickell Harris

Biology 2107K Lab

Bradford Assay

July 6, 2016

Abstract: The goal of this experiment to familiarize student with the use of Coomassie Dye the

Bradford Assay and why it is use for and its imports as well as how commercially people use it.

By the end of this experiment, finding protein concentration will be easier as a

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spectrophotometer is use to calibrate the sample to show the difference in protein concentration

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of each sample by giving the optical density of each.

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Introduction: The Bradford Assay is a type a procedure use among many other available
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procedures to determine the concentration of protein in an available sample of solution. The
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Bradford Assay uses a dye called Coomassie Brilliant Blue that binds to proteins (Bradford,
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2001). The dye and protein are usually placed in an acidic solution. The ligands on the dye bind

to the positive charges on the protein. The number of ligands that is bound is proportional to the
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number of positive charges on the protein. A Coomassie Brilliant Blue Dye normally appear as
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reddish-brown but changes color to blue in the presence of protein and the more protein there
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are, the deeper or darker the blue. The Coomassie Brilliant Blue Dye appear reddish-brown in its
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normal condition because it exists in the reagent as an unstable cation, thereby allowing it to be

an effective reagent for this assay because it binds to protein in the solution and becomes a
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stable, unprotonated form resulting in a brilliant blue color. The Bradford assay has become the

preferred method for quantifying protein in many laboratories. This spectroscopic technique is

simpler, faster, and more sensitive than the Lowry method. Moreover, when compared with the

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 Lowry method, it is subject to less interference by common reagents and non-protein

components of biological samples. Because the peak absorbance of the Coomassie blue dye in a

protein containing solution is at a wavelength of 595nm, so the spectrophotometers was set to

595nm for this experiment in order to ensure the result obtain were the most accurate results

possible.

Materials and Methodology: This experiment was done using 8 different cuvettes. After

obtaining the 8 cuvettes, 7 of them were labeled in order from 1 to 7 and the last remained

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unlabeled or was label blank by some groups. To begin, 1mL of dye was added to the cuvette

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label blank along with 20mL of the unknown. Next, 20 microliters were added to the cuvettes

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label 1-7 each. Each with a different concentration from 0.125mg/mL to 2.000mg/mL along with
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the dye too. Next, each cuvettes were covered with parafilm and inverted to mix. Then the
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samples were incubated at room temperature for a period of approximately 5 minutes plus but no
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longer than 60 minutes. After this step, the spectrophotometer was turned on and set at a

wavelength of 595nm and the cuvette with the blank sample was then placed into the
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spectrophotometer and then the “blank” button was pressed to calibrate the blank sample and the
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absorbance (OD) or the sample which was 0.000 almost always was recorded. After, the cuvette
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containing the “blank” was removed and the cuvettes labels 1 to 7 were then inserted into the
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spectrophotometer in order and each were calibrated and their resulting Optical Density (OD)

were recorded. The cuvette labeled “blank” was also reinserted into the spectrophotometer and
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recalibrated before any of the label ones were inserted into the spectrophotometer. Once the

measurements of absorbance were recorded for all the label cuvettes, a graph using standard

curve was can generated using the information of the optical density and the concentrations of

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 the samples. After plotting the points on the standard curve and getting the results, the results

from the experiment were then compared to those of the commercially available products.

Results:

Label Concentration (mg/mL) Absorbance (nm)

Blank 1XPBS OD
1 0.125 0.019
2 0.250 0.059
3 0.500 0.061
4 0.750 0.305
5 1.000 0.338
6 1.500 0.550

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7 2.000 0.685

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Discussion: Due to not getting the desired results, the experiments had to be redone repeatedly,

causing the calibration on multiple occasions so in order to get the results we desire.
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Conclusion: Since our OD wasn’t over the 1.0 limit I think we did almost an accurate result.
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References:
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1. http://www.scribd.com/doc/49138231/Bradford-Protein-Concentration-Assay-Formal-
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Report
2. Zuo, S.-S. and Lundahl, P. (2000). Analyt. Biochem. A micro-Bradford membrane protein
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assay. 284, 162–164.

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3. Krohn, R.I. (2001). The Colorimetric Determination of Total Protein, Current Protocols
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in Food Analytical Chemistry , B1.1.1-B1.1.27, John Wiley & Sons, Inc.-

4. Hopkins, (1993) Maton et. Al. Human Biology and Health. Englewood Cliffs, Michigan,

USA: Prentice Hall

5. Biology 2107K Lab Manual

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