Bovine serum albumin (BSA)
- A Biuret reagent, consisting of 2.25 gm sodium potassium tartrate, 0.75 gm Copper sulphate x 5 H2O, 1.25 gm potassium iodide was prepared, all dissolved in order in 100 mL 0.2 M NaOH (0.8 gm/100 mL). The volume was brought to 250 mL with distilled water. If a black precipitate forms, then it was discarded.
- A serial dilution of 2, 4, 6, 8, and 10 mg/mL was prepared by using a stock solution of bovine serum albumin (10 mg/mL). (Final volume for each tune is 1 mL).
- 9 mL Biuret reagent to each tube was added, immediately vortexed, and let stood at room temperature for 20 minutes.
- 1 mL of the sample labelled UNKNOWN was pipette to a test tube and step 3 was repeated.
- The absorbance was read at 550 nm using spectrophotometer.
- A standard curve of absorbance versus micrograms protein (or vice versa) was prepared, and the concentration of the unknown samples was determined from the curve. A graph of absorbance vs concentration was plotted.
Table 1: Absorbance rate of BSA at different concentration.
The absorbance rate of the UNKNWON sample determined through spectrophotometer has the value of 0.139 nm.
From the graph of Absorbance, A550 (nm) against Concentration of BSA (mg/mol),
The concentration of the UNKNWON sample is 5.3 mg/mol.
The value can be assumed approximately to 5 mg/mol.
The biuret reagent was used for both qualitative and quantitative analysis of protein. The biuret method depends on the presence of peptides bonds in proteins. The solution of proteins is treated with cupric ions (Cu2+) in a moderately alkaline medium, a purple coloured Cu2+ - peptide complex is formed that was measured quantitatively by spectrophotometer. So, biuret reagent is alkaline copper sulphate solution. The intensity of the colour produced is proportional to the number of peptide bonds that are reacting, and therefore to the number of protein molecules present in the reaction system. The reaction don't occur with amino acids because the absence of peptide bonds, and also that with di-peptide because presence of only one peptide bond, but do with tri-, oligo-, and poly-peptides. Biuret reaction needs presence of at least two peptide bonds in a molecule. The reaction occurs with any compound containing at least two bonds of: -HN-CO- , -HN-CH2- , -HN-CS- .
Because proteins differ in their amino acid compositions, each one responds somewhat differently in each type of protein assay. Therefore, the best choice for a reference standard is a purified, known concentration of the most abundant protein in the samples. This is usually not possible to achieve, and it is seldom convenient or necessary. If a highly purified version of the protein of interest is not available or it is too expensive to use as the standard, the alternative is to choose a protein that will produce a very similar colour response curve in the selected protein assay method and is readily available to any laboratory at any time. Generally, bovine serum albumin (BSA) works well for a protein standard because it is widely available in high purity and relatively inexpensive.
A standard curve is a quantitative research tool, a method of plotting assay data that is used to determine the concentration of a substance like protein. The biuret assay was performed with various known concentrations of bovine serum albumin. The response was the absorbance of the coloured product. These data were used to make a standard curve, concentration of bovine serum albumin (BSA) on the X axis and the absorbance on the Y axis. Also perform the biuret assay with your sample. You want to know the concentration of the UNKNWON in sample. A best fit line was drawn through the standards to analyze the data. For the UNKNOWN sample, the graph from the spot on the Y-axis that corresponds to absorbance of the sample was read across until it intersects the standard curve and was read down the graph until it intersects the X-axis. The concentration of UNKNWON in the sample is the value on the X-axis.
Protein in solution absorbs UV light at a wavelength of 280 nm, due to the presence of aromatic amino acids, mainly phenylalanine, tyrosine, proline and tryptophan. This property is the basis of this method. Many other biochemicals absorb near this wavelength, making an accurate quantitation difficult. If the buffer used to dissolve the protein has a high absorbance relative to that of water, there is an interfering substance in the buffer. This interference can be compensated for when the spectrophotometer is adjusted to zero buffer absorbance. Furthermore, at low concentrations protein can be absorbed onto the cuvette, thereby reducing the content in solution. Another interfering factor would be ratio of amino acids presents in different proteins which absorb at different rate as well.
Next is Lowry assay method, is based on the reduction by protein of the phosphomolybdic-tungstic mixed acid chromogen in the Folin-Ciocalteu’s phenol reagent, resulting in an absorbance maximum at 750 nm. The Folin-Ciocalteu’s phenol reagent reacts primarily with tyrosine residues in the protein, which can lead to variation in the response of the assay to different proteins. The method is sensitive to interfering substances; a procedure for precipitation of the protein from the test specimen need be to used. Most interfering substances cause a lower colour yield. However, some detergents cause a slight increase in colour. A high salt concentration can cause precipitation to form.
The Bradford assay, is based on the absorption shift from 470 nm to 595 nm observed when the brilliant blue G-250 dye binds to protein. The Coomassie Brilliant Blue G-250 dye binds most readily to arginyl and lysyl residues in the protein, which can lead to variation in the response of the assay to different proteins. Slow precipitation of the dye will occur during storage of the reagent. Therefore, the reagent must be filtered before use. The relationship of absorbance to protein concentration is nonlinear. However, if the standard curve concentration range is sufficiently small, it will approach linearity.
Conclusion: Biuret protein assay is a suitable method for measurement of protein solutions concentration.
- Biuret Protein Assay. (n.d.). Retrieved July 9, 2012, from Bioscience Excellence : http://www.gbiosciences.com/EducationalUploads/EducationalProductIMGFile/633453707995878750.pdf.
- Chemistry of Protein Assays. (n.d.). Retrieved July 9, 2012, from Thermo Fisher Scientific: http://www.piercenet.com/browse.cfm?fldID=876562B0-5056-8A76-4E0C-B764EAB3A339
- Overview of Protein Assay Methods. (n.d.). Retrieved July 9, 2012, from Thermo Fisher Scientific : http://www.piercenet.com/browse.cfm?fldID=BE219700-9B95-43A1-A3DA-83800F1A0392
- Protein Assays. (2006, September 6). Retrieved July 9, 2012, from Experimental Bioscience: http://www.ruf.rice.edu/~bioslabs/methods/protein/protein.html
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***** A thorough consideration of the use of the biuret test for protein assays. Good use of A level scientific language throughout.