In this practical exercise you will be using biuret reagent. Why is biuret reagent used in this practical? The biuret reagent is used to assess the concentration of the protein because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the colour, and therefore the absorption, is directly proportional to the protein concentration. Q 2. What is the experimental reason for constructing a standard curve?
Multiple samples with known properties can be measured and graphed, which then enables the same properties to be determined for the unknown samples, in this case the concentration, by interpolating the graph which depicts the relationship between the absorbance and concentration. Q 3. Based on the above information (in the prac manual) and your own thinking, which plant do you hypothesize will have more rubisco, one raised in the sun or one raised in shade? It is hypothesised that the sun raised silverbeet plant will have more rubisco than the shaded silverbeet plant.
Should the line connect all the data points or should it be a line of best fit? Why? (2 sentences) A line of best fit as it will give an accurate representation of the relationship between absorbance and concentration between the different samples. The more samples used, the closer the results would be to the line of best fit. Q 5. Is it possible to determine the protein concentration of a solution from the standard curve if the measured absorbance is off the scale of the y-axis, i. e. higher than the highest y-value on the standard curve? Give your reasons.
It’s not possible to determine the protein concentration because the data gathered from the standard solutions is in a certain range (specific scale). Therefore the relationship between absorbance and concentration cannot be determined for anything outside of this range. Q 6. Describe two ways in which you would modify the experimental procedure to determine the protein concentration of this solution? Give full details of the two experimental procedures. (2 and 4 sentences) One way in which the experimental procedure could be modified is diluting the sample.
This involves decreasing the volume of BSA Protein solution and increasing the buffer volume by the same amount. As a result the absorbance would be lower and fit on the graph. Another way may be to extend the curve by increasing the concentration of the BSA Protein solution which would provide a greater range. Q 7. Do your results support your hypothesis regarding these tissue samples? (2-3 sentences) The hypothesis regarding the tissue samples was supported. The absorbance of the sun raised silverbeet plant was found to be 0. 801, which was greater than the shaded silverbeet plant with an absorbance of 0.
One possible reason for the results not supporting your experimental hypothesis is experimental error. List three examples of possible sources of error that may have occurred in your work in this practical class. (2-3 sentences) A possible error may be adding inaccurate amounts of buffer between samples, causing them to dilute either excessively or insufficiently, resulting in an inaccurate standard curve. Another error could be leaving the sample solutions for over 25 minutes, which could cause the protein to start degrading.
Not zeroing the spectrophotometer or cleaning the tube properly can affect the absorbance value. Q 9. Even if your results do support the experimental hypothesis, give three major possible explanations for a negative result (i. e. biological reasons – not experimental errors). (4-5 sentences) Biological errors involve using different parts the plant, such as a stem or leaf and one may have a higher or lower absorbance than the other. Another reason may be that different aged samples were used, such as a baby plant which may have a higher absorbance compared to an aging plant.
Furthermore, the sun raised silverbeet plant could have had excessive exposure to the sun and was beginning to die, causing a lower absorbance. Q10. What is the single major design problem in this experiment in drawing conclusions about different concentrations of rubisco in response to different growth light conditions? (2 sentences) Rubisco is not the only protein in the plant as it makes up 80% of the total protein in plant leaves. This means that there is 20% of other existing proteins that may have contributed to the results.