User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/09/23

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Objective

The objective for today's lab work is to create a series of lysozyme solutions and then measure their absorbances using a Bradford assay.

Protocol

We based our protocol for our Bradford Assay off of the one that Dr. Hartings outlined in his lab notebook. However, we did not follow his protocol exactly; ours is outlined below.

  1. First, we made a stock solution of lysozyme in Tris buffer:
    1. The Tris buffer had already been made by another group and was in the hood. It was a 50mM Tris + 50mM NaCl buffer with a pH of 7.5.
    2. Our goal was to make a lysozyme stock solution that was 0.0100g in 5mL buffer. However, when measuring out the lysozyme, we measured out 0.01039g instead. We diluted this mass of lysozyme to a final volume of 5mL in buffer using a 5mL volumetric flask.
    3. Thus, our goal was to make a 2000µg/mL stock solution of lysozyme in buffer, but we actually made a 2078µg/mL stock solution.
  2. Next, we made another stock solution of lysozyme in Tris buffer using the stock that we made in Step 1:
    1. First, we calculated how much of our stock solution from step 1 we would need in order to make 50mL of a 50µg/mL solution of lysozyme in buffer.
      1. Calculations:
        M1V1=M2V2
        where M1=concentration of the 5mL stock solution = 2078µg/mL
        V1=volume of the 5mL stock solution we would need
        M2=final concentration of the new stock solution we wanted to make=50µg/mL
        V2=final volume of the new stock solution we wanted to make=50mL
        We solved for V1 and got 1.2031mL.
    2. We pipetted 1.2031mL of the stock solution into a 50mL volumetric flask and diluted the solution to a final volume of 50mL in buffer. This gave us 50mL of a 50µg/mL stock solution of lysozyme in buffer.
  3. Next, we prepared samples in cuvettes for our Bradford Assay. The table below shows the contents of each of the 12 cuvettes that we prepared.
Table 1: Contents of Each of the Cuvettes in the Bradford Assay
Cuvette # Concentration (µg/mL) Volume of 50µg/mL Stock (mL) Volume of Bio-Rad Protein Assay Reagent (mL) Volume of Buffer (mL) Total Volume (mL)
11.000.0600.6002.3403.000
21.500.0900.6002.3103.000
32.000.1200.6002.2803.000
42.500.1500.6002.2503.000
53.000.1800.6002.2203.000
64.000.2400.6002.1603.000
75.000.3000.6002.1003.000
86.000.3600.6002.0403.000
97.000.4200.6001.9803.000
108.000.4800.6001.9203.000
119.000.5400.6001.8603.000
1210.000.6000.6001.8003.000

In the table, the first column shows the number of the cuvette. We numbered the cuvette from 1 to 12 in order of increasing concentration of lysozyme. The second column shows the concentration of lysozyme in the cuvette. We arbitrarily assigned a concentration, between 1 and 10µg/mL of lysozyme in buffer, for each of the cuvettes. The third column shows the volume of the 50µg/mL stock solution of lysozyme, which we made in step 2.2, that we needed to pipette into the cuvette in order to get the final concentration that we assigned that cuvette. The fourth column shows the volume of the Bio-Rad Protein Assay reagent that we added to each cuvette. We pipetted 600µL, or 0.600mL, into each cuvette. The fifth column shows the volume of the buffer that we added to the cuvette in order to adjust the concentration and final volume of the cuvette. The last column shows the final volume of solution in each cuvette, which was always 3mL.

In order to determine the volume of the 50µg/mL stock solution that we needed in each cuvette (column #3 in the table), we did the following calculation:

Let
Ma=concentration of the 50µg/mL stock solution=50µg/mL
Va=volume of stock solution needed in the pipette
Mb=concentration of lysozyme wanted in the cuvette (these are the values in column #2 of the table)
Vb=total volume of solution in cuvette=3mL
Vr=volume of Bio-Rad Protein Assay Reagent in cuvette=0.600mL
Vf=volume of buffer added to each cuvette


For cuvette #1, Mb=1µg/mL. Then,

MaVa=MbVb

Va=(MbVb)/Ma

Va=((1µg/mL)(3mL))/(50µg/mL)

Va=0.060mL


Vf=Vb-Va-Vr

Vf=(3mL)-(0.060mL)-(0.600mL)

Vf=2.340mL


4. We then measured the absorbance of a blank (a cuvette filled with 3mL of the Tris buffer) and each of the cuvette samples using the UV-Vis Spectrometer. The wavelength range that we took measurements over was 800-400nm.

Analysis

Figure 1: Absorbance of Each Lysozyme Sample in the Bradford Assay as a Function of the Wavelength of Indecent Light (nm)


The figure above shows the raw data from the Bradford Assay. It shows the absorbance of the blank and of each lysozyme sample as a function of the wavelength of incident light. Each different colored line in the plot represents a different lysozyme sample; the concentration of the samples are listed in the legend on the right hand side of the graph. These absorbance values are not corrected for noise or the blank.


Figure 2: Corrected Absorbance of Each Lysozyme Sample in the Bradford Assay as a Function of the Wavelength of Indecent Light (nm)


The figure above shows the corrected absorbance of each lysozyme sample in the Bradford Assay as a function of the wavelength of incident light. The absorbance values were corrected by first subtracting the absorbance value of the blank from the absorbance value of each sample for every wavelength. Then, the absorbance value at the isobestic point for each sample was subtracted from all the absorbance values for that sample. The isobestic point was at 535nm. Each different colored line in the plot represents a different lysozyme sample; the concentration of the samples are listed in the legend on the right hand side of the graph.


Figure 3: Calibration Curve: Absorbance of Lysozyme at of Incident Light of Wavelength 600nm as a Function of Concentration (µg/mL)

The figure above is the calibration curve for the Bradford Assay. It shows the absorbance of lysozyme as a function of concentration of lysozyme when the indecent light has a wavelength of 600nm. The absorbance values are taken from the corrected absorbance calculated for all of the lysozyme samples from the assay. The wavelength of incident light of 600nm was chosen for the calibration curve because this wavelength is the wavelength at which the lysozyme samples showed peak absorbance.

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