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Determination of various protein concentrations by means of spectrophotometry

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  1. Introduction
  2. Methods
  3. Results
  4. Sample calculations
    1. Standardized concentration calculations
    2. Lowry Assay concentration calculations
    3. UV Direct Assay concentration calculations
  5. Discussion
  6. Conclusion
  7. References

Spectrophotometry relies on the varying capabilities of molecules to absorb photons of light. This absorbance is characteristic of specific compounds, and can therefore be utilized to quantify and classify the presence/quantities of various compounds. (Harris, 2007) With the use of standard calibration curves, the concentrations of various samples can be graphically interpreted as a function of the absorbance. The relation between absorbance and concentration is governed by the Beer-Lambert law, which states that the absorbance changes proportionally with the concentration. (Harris, 2007) This fundamental principle allows for a direct correlation between the examined absorbances and the corresponding concentrations. Some samples require preparation prior to direct examination, as they may be ?invisible? to the spectrophotometer initially. These colorimetric analysis techniques utilize a dying reagent, which acts to increase the absorbance of the sample. (Congdon, 1993)

[...] In order to determine the concentrations of various proteins, this investigation utilized several assays, as a method of both preparing and analyzing protein samples. The Coomassie Blue assay relies on the Colorimetric interactions, between the additional compounds and the protein analyte. This technique allows samples with little or no absorptivity to become ?visible? either by chemically altering the component, or producing alternate compounds in proportional quantities. (Congdon, 1993) This highly utilized technique is often employed for its ease of use, as well as its high sensitivity to analyte. [...]

[...] This discrepancy may be explained simply by a matter of elapsed reaction time. Colorimetric assays utilize the reactivity of components to essentially reveal a compound in solution. If this reaction has not run to completion, the values obtained may be inaccurate, as lower concentrations require much more time to react. (Gore, 2000) This condition may account for the inefficiencies observed, but may also be related specifically to poor sampling practices and the inclusion of foreign compounds. Variations within the path length seemed to follow a similar trend, as an increased path length should yield an increased molar absorptivity. [...]

[...] (Table 02) In order to determine the relative concentrations of each protein (BSA, Gamma Globulin, Hemoglobin) several analytical techniques were employed as standardization mechanisms. The Coomassie Blue assay was utilized to generate two stand curves, which formed a fairly linear relation between the absorbance and concentration. (Figure 02) An alternate colorimetric method was utilized to generate a similar distribution, although it employed the procedures of the Lowry assay. (Figure 03) The last protein assay did not involve any direct sample preparation, as it relied simply on the absorption of UV light. [...]

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