1: Can you reliably use the standard curve you obtained, for the samples 5 and 6
ID: 532012 • Letter: 1
Question
1: Can you reliably use the standard curve you obtained, for the samples 5 and 6? Why yes / why no? If no, what can you do to avoid making a new calibration curve?
2: If you do need to make a new calibration curve, what range of BSA concentrations would you use?
3: Your protein P concentration measured with BSA as a standard was 0.5mg/ml. If you used -globulin as a standard for the same assay, would the protein P concentration measure higher or lower? Approximately how much higher / how much lower?
0.601 y = 0.0508x + 0.0077 R2-0.9997 0.50 0.40 0.30 0.20 0.10 0.00 r1HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 10 H1 +6 4 4321Explanation / Answer
1. No. The useful range of absorbance scale is from 0-2 (the % of transmitted light at absorbance 2 is 1%) but it is desirable to keep within the range 0-1. Above 1, the results become unbelievable due to scattering of light. In the present case, the absorbance of samples 5 and 6 are 2.47 and 1.87 respectively. Thus if you are preparing new calibration curve also, the results will not be much reliable.
The present calibration curve is only up to absorbance 0.5, thus any absorbance above this value cannot take for finding the unknown concentration.
Using the same calibration curve itself, we can find the concentrations of the unknown samples by diluting the samples. For eg. Sample 30 microliter of sample 5 and 6 are diluted to 150 microliters (5 times) and measure the absorbance. Find the corresponding concentration from the calibration curve. To obtain the exact concentration multiple the obtained concentrations with 5.
2. This is not a reliable method. Because if we are preparing a solution of higher concentrations the absorbance will go above 1. However, theoretically, it is possible. Prepare 300 mg/mL stock solution. From this take 0, 10,15,20,30 and 60 microliters and prepare the solution by adding the remaining amount of water to make it to 150 microliters and perform the experiment. (No the concentration is increased by 6 times, ie for final concentration, absorbance will come around 3)
3. BSA has 24, phenylalanine, 2 tryptophan and 20 tyrosine aminoacids. Whereas gamma globulin has 12 phenylalanine, 5 tryptophan and 1 tyrosine. Since the aromatic aminoacds contributes towards the absorption after 250 nm, we have to use much higher (2.5 times) concentration of gamma globulin.
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