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  • 1. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 PRACTICAL : Spectrophotometry; Quantitative Determination of Protein 3 (**Please bring along graph papers for this practical) Introduction The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. It is subjective, i.e., dependent on the amino acid composition of the measured protein. The Bradford protein assay was developed by Marion M. Bradford. The Bradford assay, a colorimetric protein assay, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its bluer form to bind to the protein being assayed. During the formation of this complex, two types of bond interaction take place: the red form of Coomassie dye first donates its free electron to the ionizable groups on the protein, which causes a disruption of the protein's native state, consequently exposing its hydrophobic pockets. These pockets on the protein's tertiary structure bind non- covalently to the non-polar region of the dye via van der Waals forces, positioning the positive amine groups in proximity with the negative charge of the dye. The bond is further strengthened by the ionic interaction between the two. The binding of the protein stabilizes the blue form of the Coomassie dye; thus the amount of the complex present in solution is a measure for the protein concentration, and can be estimated by use of an absorbance reading. The (bound) form of the dye has an absorption spectrum maximum historically held to be at 595 nm. The cationic (unbound) forms are green or red. The binding of the dye to the protein stabilizes the blue anionic form. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample. Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemicals that may be present in protein samples. An exception of note is elevated concentrations of detergent. Sodium dodecyl sulfate (SDS), a common detergent, may be found in protein extracts because it is used to lyse cells by disrupting the membrane lipid bilayer. While other detergents interfere with the assay at high concentration, the interference caused by SDS is of two different modes, and each occurs at a different concentration. When SDS concentrations are below critical micelle concentration (known as CMC, 0.00333%W/V to 0.0667%) in a Coomassie dye solution, Page 1 of 5
  • 2. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 the detergent tends to bind strongly with the protein, inhibiting the protein binding sites for the dye reagent. This can cause underestimations of protein concentration in solution. When SDS concentrations are above CMC, the detergent associates strongly with the green form of the Coomassie dye, causing the equilibrium to shift, thereby producing more of the blue form. This causes an increase in the absorbance at 595 nm independent of protein presence. Materials, reagents and equipments: Samples: low fat milk, soy milk and cultured-drink Dye reagent, Bovine γ-globulin standard set, 1X Phosphate buffered saline (PBS) Spectrophotometer Cuvettes Microtubes 100 - 1000 ul adjustable-volume micropipette 10-100 ul adjustable-volume micropipette 2 – 20 ul adjustable-volume micropipette Methods I) Construction of linear graph from known protein standards 1. Get the set of data from your tutor and fill in the Table 1 as below. 2. Construct a standard curve linear by plotting the A595 values of the known standards against concentration from the data in table below. Table 1 Standard Curve Absorbance Values Standard Standard (mg/ml) A595 #1 0.125 0.548 #2 0.250 0.010 #3 0.500 0.703 #4 0.750 0.897 #5 1.000 1.048 #6 1.500 1.222 #7 2.000 1.138 II) Quantitative determination of protein 1. Prepare a 1:50 dilution of the protein sample using PBS in two microtubes and labeled them with Sample A, Sample B and Sample C. 2. Label 4 cuvettes as follows: Label Standard (mg/ml) Page 2 of 5
  • 3. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 Blank PBS A Sample A B Sample B C Sample C 3. Add 1 ml of dye reagent to each cuvette and 20 µl of PBS to the cuvette labeled blank. 4. By using a fresh tip for each sample, pipette 20 µl of each standard into the diluted sample into the appropriate cuvette. 5. Cover each cuvette with parafilm and invert each cuvette 3 times to mix. 6. Incubate at room temperature for a period of at least 5 minutes (but not to exceed 60 minutes). 7. Visually compare the color of your unknown samples against the standards of known concentrations. A representative set of standards and a typical color spectrum are shown in figure below. Using the palette of standards, try to qualitatively determine to which known standard your unknown sample corresponds. • Examine the color of the first unknown • Compare it to Std. #1 • Is it lighter or darker? • Compare it to Std. #2 • Is it lighter or darker, etc. • Record your observations in Table 2 below. Table 2 Unknown protein concentration Sample Estimated Protein Concentration (mg/ml) A 0.250 B 1.500 C 1.000 Page 3 of 5
  • 4. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 8. Getting data from spectrophotometer and insert the absorbance data and the concentration values for the unknown samples in Table 3. Read absorbance of samples on spectrophotometer within one hour. Table 3 Spectrophotometric Data for Unknown Samples Sample A595 Protein Concentration (mg/ml) A 0.681 B 1.008 C 0.950 9. Compare your quantitative spectrophotometer determined concentrations with qualitative estimates from table 3 to the table below. (Table 4) Table 4 Estimated vs Quantitated Protein Concentrations Sample Estimated Protein Concentration (mg/ml) – from table 2 Quantitated Spectrophotometer Protein (mg/ml) –from table 3 A B C 10. Read the concentration of unknown samples by reading across from the absorbance of unknown samples until meet the intersection with the standard curve and then read the concentration. Record these data in table 5. 11. Adjust the final concentration of the unknown samples determined from spectrophotometer before by multiplying the concentration by the dilution factor used. For example, sample A is diluted 1:50 gives a reading of 0.224 absorbance units, which gives a concentration of M mg/ml. The final concentration of milk is M x 50 = ___________mg/ml Table 5 Final concentration of unknown samples Sample A595 Concentration read from standard curve (mg/ml) Dilution Factor Final Concentration (mg/ml) A B C 12. Find the protein content information for your samples on the package label (look for ‘Nutrition Facts’). Convert this information into mg/ml of protein; compare the values from the standard curve. Fill in table 5 below. Page 4 of 5
  • 5. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 Table 6 Comparing measured protein concentrations to the values found on food labels Sample Bradford Assay (mg/ml) Final Concentration from Table 5 (mg/ml) Food Label (mg/ml) A B C Questions: 1. What are the functions of spectrophotometer? spectrophotometer is a machine which tells about the growth of microorganisms in culture by examining the colour change. 2. Why might the values you get be different from those of the manufacturer? 3. List the major sources of errors you can observe in this experiment. A high concentration of buffer will cause an overestimated protein concentration due to depletion of free protons from the solution by conjugate base from the buffer. This will not be a problem if a low concentration of protein (subsequently the buffer) is used. 4. How long can the samples sit before being read? 2 minute or less. 5. Do bubbles on the surface of the samples affect reading? Yes, that’s why we put it carefully. 6. Do fingerprints on the cuvette affect the reading? Yes, that’s why we wipe it before we put it. Page 5 of 5
  • 6. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 Table 6 Comparing measured protein concentrations to the values found on food labels Sample Bradford Assay (mg/ml) Final Concentration from Table 5 (mg/ml) Food Label (mg/ml) A B C Questions: 1. What are the functions of spectrophotometer? spectrophotometer is a machine which tells about the growth of microorganisms in culture by examining the colour change. 2. Why might the values you get be different from those of the manufacturer? 3. List the major sources of errors you can observe in this experiment. A high concentration of buffer will cause an overestimated protein concentration due to depletion of free protons from the solution by conjugate base from the buffer. This will not be a problem if a low concentration of protein (subsequently the buffer) is used. 4. How long can the samples sit before being read? 2 minute or less. 5. Do bubbles on the surface of the samples affect reading? Yes, that’s why we put it carefully. 6. Do fingerprints on the cuvette affect the reading? Yes, that’s why we wipe it before we put it. Page 5 of 5