Objective
Today's objective was to create a series of lysozyme solutions and measure their absorbance with the Bradford assay to create a calibration curve.
Protocol
The Bradford Assay was conducted based on Dr. Hartings' outlined instructions in his lab notebook. Some modifications to the procedure were made and are shown below.
- First a high concentration lysozyme stock solution was created by adding 0.0100g in 5mL Tris buffer.
- Tris buffer was made previously by another group and composed of 50mM Tris and 50mM NaCl buffer with a corresponding pH= 7.5.
- In reality, what was meant to be a 2000ug/mL stock solution turned out to be 2078ug/mL stock lysozyme solution.
- Second, the first 2078ug/mL stock lysozyme solution was diluted to 50ug/mL
- This was performed by using M1V1=M2V2 where M1 = 2078ug/ml, V1 = volume of initial stock needed, M2 =50ug/mL and V2 = final volume of new stock solution of 50mL
- V1= 1.2031mL of 2078ug/mL solution in a total of 50mL of buffer gives us 50mL of a 50ug/ml solution of lysozyme.
- Thus, 1,203.1uL of the initial lysozyme stock was pipetted into a 50mL volumetric flask which was then filled up the 50mL meniscus.
Bradford Assay Sample Prep
- The bradford assay requires samples to be prepared with an assay reagent, buffer and protein according to Dr. Hartings' instructions in his lab notebook
- The table below shows the contents of the lysozyme samples prepared for the Bradford assay along with their respective concentrations of lysozyme. The samples are in order of increasing lysozyme concentration ranging from 1ug/mL to 10ug/mL.
- The volume of 50ug/mL lysozyme solution added to each cuvette is based on the target lysozyme concentration in that cuvette and was calculated using M1V1=M2V2 with M2 being the target concentration, V2 = 3mL, V1= volume of 50ug/mL stock solution added and M1=50ug/mL.
- These samples were then allowed to incubate at least 5 minutes after mixing and then analyzed under UV-Vis over a 400-800nm wavelength range. An absorbance for a blank with only the buffer was also taken and factored into data analysis.
| Sample #
|
Lysozyme Concentration (µg/mL)
|
Volume of 50µg/mL Lysozyme Stock (mL)
|
Volume of Bradford Protein Assay Reagent (mL)
|
Buffer Volume (mL)
|
Total Volume (mL)
|
| 1 |
1 |
0.06 |
0.6 |
2.34 |
3
|
| 2 |
1.5 |
0.09 |
0.6 |
2.31 |
3
|
| 3 |
2 |
0.12 |
0.6 |
2.28 |
3
|
| 4 |
2.5 |
0.15 |
0.6 |
2.25 |
3
|
| 5 |
3 |
0.18 |
0.6 |
2.22 |
3
|
| 6 |
4 |
0.24 |
0.6 |
2.16 |
3
|
| 7 |
5 |
0.3 |
0.6 |
2.1 |
3
|
| 8 |
6 |
0.36 |
0.6 |
2.04 |
3
|
| 9 |
7 |
0.42 |
0.6 |
1.98 |
3
|
| 10 |
8 |
0.48 |
0.6 |
1.92 |
3
|
| 11 |
9 |
0.54 |
0.6 |
1.86 |
3
|
| 12 |
10 |
0.6 |
0.6 |
1.8 |
3
|
Data
Raw absorbance vs. Wavelength data was first corrected for the blank (buffer and Bradford protein reagent only) by subtracting the blanks spectra from all other spectra for each concentration. Then absorbance at the isosbestic point for each spectra (535nm) was subtracted from that specific concentration's spectra. This data was used to calculate the graphs below, first using only the absorbance data from the absorbance peak wavelength of 600nm and then showing the full corrected absorbance data.
|
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