Chemistry Essay on Biuret Protein Assay

AbstractThe use of Lowry assay and Biuret assay are necessary for conducting the experiment to establish the protein concentration. The Biuret test is a chemical test used in distinguishing the peptide bonds presence. IN the presence of the peptides, the violet-colored coordination appears in a copper (ii) ion complexes of an alkaline solution. Researchers have come up with some alternatives to the text, for example, Modified Lowry test and BCA test. The Biuret reaction applies in assessing the protein concentration, as per Beer-Lambert law. Regardless of its name, the reagents used are neither containing biuret ((H2N-CO-) 2NH). The test is given that name since it also produces a positive reaction to the peptide-like bonds in the biuret molecule.

Lab Report on Determining the Protein Concentration

Introduction

The laboratory approach for determining the precise protein quantity in a solution is often necessary for the field of biochemistry, and there are various techniques for measuring protein concentration. Protein assay is the process meant to measure the complete protein in the solution. This technique is quantitative if the protein tested is present in sufficient volume in that it is possible to plot a standard curve. If it is impossible to attain this, the normal protein like albumin may be suitable for a standard curve with the consideration that the result of an unknown protein is possibly semi-quantitative. Since many proteins are in small quantities rather than in large quantities, the standard curves for the protein assays are normally founded on the use of either bovine gamma globulin (IgB) or bovine serum albumin (BSA). According to Kruger (1994), the Bradford protein assay is the technique of determining the concentration of protein in the mixture. The technique operates on the proportional binding of the dye Coomassie to protein. The higher the protein content, the same way there will be more of the Coomassie binds, consequently producing a substantial color change in the mixture. The Beer-Lambert Law states that a standard curve of the absorbance against the concentration of protein (derived from the measure of the absorbance of the protein solution of a known concentration) is applied in estimating the concentration of protein together. On the other hand, it is also sensitive to the non-protein sources, specifically detergents, eventually becoming the nonlinear having a higher protein concentration (Kuaye, 1994).

The spectrophotometer

The spectrophotometer (chromogenic) methods, basically involve, concentrates on the measure of the colored product that is produced by the organic molecule together with the protein. Also, the concentration of protein can be established from the proteins self (intrinsic) UV absorbance. It is imperative to note that, although, these approaches may not give similar results due to different proteins of equal concentration. Additionally, various methods can provide somehow different results; though, from the same protein. It is identified that there is no complete photometric protein concentration assay. All the techniques have both advantages and disadvantages; therefore there is need to select one by considering the following aspects: specificity, sensitivity, specificity, the quantifiable concentration range, protein nature, accuracy, presence of impurities interfering with the results as well as the time available for the appraisal (Kuaye, 1994).

Measuring Protein Concentration through Absorption Spectrophotometer

This particular experiment will demonstrate how to homogenize a tissue to the specific protein, together with how to utilize the protein assay agent to get the protein concentration in the sample. Absorption spectrophotometry is a method for defining the concentration of protein substrate in the solution. In the experiment, it will be identified that the dissolved protein substrate will absorb light of particular wavelengths typical of that of the substance (Watters, 1978). The samples arranged in the tubes, then when the light of those wavelengths goes through the samples, and then the solute will absorb some of the light, minimizing the degree of light that passes through the sample. In this case, a spectrophotometer will be used in measuring the change in the magnitude of the light that enters and leaves the sample. The light that penetrates through the solute (not absorbed) is referred to as transmitted light. On the other hand, absorbance refers to the variance in the original and transmitted light. Absorption spectrophotometry can be applied in the examination of the properties of various biological molecules such as enzymes, pigments DNA among other much more minor organic molecules. With the same practice, various biological activities in the living cells can be measures such as the rates of photosynthesis as well as enzyme activities (Determination of protein concentration, 2017). This research is aimed at identifying the concentration of protein in a known solution by the use of the Biuret technique of a protein assay, together with learning how spectrophotometer is used in calculating the absorbance level of various protein concentrations. The technique of Biuret needs the solution to be mixed with a Biuret reagent before being passed through a spectrometer.

Method

Five test tubes of 0 mg/ml, 2 mg/ml, 5 mg/ml, 8 mg/ml and 10 mg/ml, are filled with different Bovine Serum Albumine (BSA) of protein concentration, and the labeled 1, 2, 3, 4, and 5 respectively.

One of the test tubes is filled with distilled water only, acting as a control experiment.

Every tube was added with 4 ml of Biuret reagent and instantaneously followed with the vortex.

The solutions were then set aside for nearly 30 minutes. In determining the absorbance of the solutions, a little amount of every solution was put in the spectrometer, and the reading for absorbance was recorded, varying between 0 to 0.7. With the use of the data from concentration together with the BSA protein absorbance data, a standard curve is concentrated, illustrating the absorbance v/s. Concentration graph.

Unknown samples were then prepared for the second part of the experiment; the samples had different concentrations with the method of 1, 1/2, 1/10 and 1/100. Biuret reagent was added to every solution and closely followed by Vortex, before being set aside for approximately 30 minutes. Subsequent, the spectrophotometer noted the rate of absorption. Lastly, the standard curve was plotted with the data derived from the first experiment hence assessing the concentration of the unknowns.

Different group members took part in the experiment

Jessica added 850 micro liters of ddH20

Rolan added 5000 micro liters of ddH20

Rolan added 3000 ddH20 into 2-4 std tubes

Jessica added 3000 added ddH20 into fifth std tube

Jessica added 150 micro liters of 2 mg/ml BSA stock solution to Std 1

Ezekiel added 3000 ml of BSA solution to 1 to 2 std tube and changed the tip

Ezekiel added 3000 ml of BSA solution to 2 to 3 labeled std tube and changed the tip

Rolan added 3000 ml of BSA solution to 3 to 4 labeled std tube and changed the tip

Rolan added 3000 ml of BSA solution to 4 to 5 labeled std tube

Jessica mixed .1 unknown with .9 H20

Rolan added dilutions (std 1-5) to 96 wells

Rolan added commassie blue to each well

1 2 3 4 5 6 7 8 9 10 11 12 A 1.239 1.219 1.131 1.014 0.806 0.175 1.189 0.046 0.046 0.046 0.045 0.053 B 1.263 1.04 1.065 1.097 0.792 0.163 1.209 0.046 0.047 0.047 0.046 0.046 C 1.228 1.174 1.169 1.076 0.85 0.178 1.239

0.047 0.046 0.047 0.046 0.046 D 0.049 0.046 0.046 0.047 0.047 0.048 0.047 0.046 0.046 0.047 0.046 0.046 E 0.046 0.046 0.054 0.046 0.046 0.047 0.047 0.048 0.047 0.046 0.046 0.046 F 0.046 0.046 0.047 0.046 0.046 0.048 0.047 0.046 0.046 0.046 0.046 0.045 G 0.047 0.048 0.047 0.046 0.046 0.046 0.047 0.047 0.047 0.046 0.047 0.045 H 0.045 0.047 0.047 0.046 0.046 0.046 0.046 0.046 0.046 0.047 0.046 0.045 50 25 12.5 6.25 3.125 A 1.064 1.044 0.956 0.839 0.631 B 1.1 0.877 0.902 0.934 0.629 C 1.05 0.996 0.991 0.898 0.672 50 25 12.5 6.25 3.125 Mean 1.243333 1.144333 1.121667 1.062333 0.816 Std Dev 0.017898 0.093115 0.052624 0.043155 0.030265 Mean of raw data - mean of blank 1.071333 0.972333 0.949667 0.890333 0.644 Mean of blank 0.172 2413028575

Conclusion and Recommendation

Proteins are composed of polymers made up of amino acid combined with peptide bonds (they absorb light, and they have partial double bonds character). The characteristics are also essential in gauging the absorbency. Biuret reagents contain Cu2 ion that shapes the protein's peptide bonds. The Biuret technique is less sensitive; though, it is the most rectilinear because its color may marginally differ with regards to the concentration of the protein. A standard curve (Absorbance v/s Concentration) was plotted with the data derived from the five samples of the identifiable concentration and absorbance. When unknown solution 1/100 together with the unknown solution 1/10 is run through the spectrometer, they are calculated to have 0.1032 and 0.5573 absorbances respectively. The values are later marked on the graph's y-axis, plotted to form a curve. The points of intersection for the concentration of protein show the unknown 1/100 as mg/ml and unknown 1/10 as mg/ml. Although, there no possible means that the unknown 1 concentration and unknown 2 concentrations could be analyzed with the standard curve, reason of this is due to the absorbance for the two unknowns are more extra as compared to that of the span of detailed absorbance of the BSA five samples (Bradford, 1976).

Decisively, as per the standard curve is drawn with the recorded data in the experiment, is evident that absorbance rate is directly proportioned to that of the protein concentration. The increase in absorbance level will consequently lead to increase of the protein concentration. Likewise, this depends on the level of water contained in the solution. Furthermore, increase in the water volume, leads to the concentration reduction, therefore decreasing the absorbance. The process of determining the concentration of the unknown protein solution will be easier when applying the standard curve plotted with the data derived from this research experiment.

Protein is an indispensable and a major class of cellular macromolecules. Therefore, determining protein fixations in an incredibly differing array of experimental settings persistently draws the attention, together with assessments the resources, of numerous investigators. A fascinating idea about this research is that the techniques used are all optical methods. This means that determination of the protein concentration depends on the optical characteristics, either turbidity or absorption of the properties solutions, hence the only device capable of measuring such optical properties is spectrophotometer (or colorimeter). Although, there are other types of methods with the ability for protein determining, such as the gravimetric, volumetric, etc. Optical techniques of biochemical study are versatile, convenient and sensitive (Bitesize Bio, 2017).

DiscussionHow to use a protein assay standard curveTypical protein assays are n...