Optional Absorbance Homework
Based on 2/13/97 Lab Data
1) The absorbance of several CAB solutions at 280 nm were measured:
| Group 1: |
stock |
1.132 |
| |
5x dilution |
0.177 |
| |
10x dilution |
0.082 |
| Group 2: |
stock |
1.090 |
| |
10x dilution |
0.087 |
Calculate the concentration of CAB in all samples
- (a)
- by using the reported extinction coefficient of 1.83 ml/mg/cm
- (b)
- by estimating the extinction coefficient from the tryptophan and tryosine content of CAB
How consistent are the absorbance readings of the stock and the diluted samples? If they don't agree, what are some reasons why not?
2) In the wavelength scan mode, absorbance peaks at 280 nm and 216 nm were found. What parts of the protein are responsible for these two peaks?
3) The following data were collected by measuring the absorbance at 400 nm as a function of time of solutions containing CAB and pNAc. In both cases the 10x dilution of CAB stock was used. 450 microliters of CAB solution was mixed with 25 microliters of 55 mM pNAc. The control is pNAc without CAB.
If you'd prefer to download the data and open it directly into an application like Excel, go here and save the page as a text file.
Absorbance at 400 nm
Time
(sec) |
Group 1 |
Group 2 |
Control |
| 0 | 0.684 | 0.610 | 0.046 |
| 6 | 0.720 | 0.684 | 0.047 |
| 12 | 0.757 | 0.758 | 0.047 |
| 18 | 0.794 | 0.830 | 0.047 |
| 24 | 0.831 | 0.899 | 0.048 |
| 30 | 0.868 | 0.964 | 0.048 |
| 36 | 0.905 | 1.027 | 0.049 |
| 42 | 0.941 | 1.092 | 0.050 |
| 48 | 0.977 | 1.155 | 0.050 |
| 54 | 1.013 | 1.216 | 0.051 |
| 60 | 1.047 | 1.276 | 0.052 |
| 66 | 1.081 | 1.336 | 0.052 |
| 72 | 1.114 | 1.394 | 0.052 |
| 78 | 1.146 | 1.450 | 0.052 |
| 84 | 1.178 | 1.503 | 0.052 |
| 90 | 1.210 | 1.554 | 0.053 |
| 96 | 1.242 | 1.604 | 0.053 |
| 102 | 1.274 | 1.654 | 0.054 |
| 108 | 1.306 | 1.703 | 0.054 |
| 114 | 1.338 | 1.748 | 0.055 |
| 120 | 1.369 | 1.792 | 0.056 |
- (a)
- Plot the data. Does the absorbance increase linearly with time? If so, why? If not, why not?
- (b)
- Are the data from the two groups consistent?
- (c)
- If you had a CAB sample at unknown concentration could you estimate the concentration by performing a pNAc assay? How?
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