Quantification Data Analysis PDF

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Quantification Data Analysis...

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Name Kelly Hewes MCB 253 - H12 22 February 2019 Quantification Data Analysis Presentation: Table 1. Measurement of Absorbance at 280 nm for varying BSA concentrations BSA Sample Number

Concentratio

Volume of BSA (mL)

Volume of PBS (mL)

A1 at 280nm

A2 at 280nm

Ax at 280nm

1 2

n (mg/mL) 0 1.5

0 0.9

1.2 0.3

0 0.973

0 0.910

0 0.9415

3

1.25

1.0 of sample 2

0.2

0.776

0.821

0.7985

4

1.00

0.96 of sample 3

0.24

0.606

0.686

0.646

5 6

0.75 0.5

0.9 of sample 4 0.8 of sample 5

0.3 0.4

0.448 0.112

0.541 0.326

0.4945 0.219

7

0.25

0.6 of sample 6

0.6

0.155

0.182

0.1685

Table 2. Measurement of Absorbance at 280 nm for Unknown Protein Unknown Dilution Sample 1 Sample 2

A1 at 280 nm 0 0.607

A2 at 280 nm 0 0.648

Ax at 280 nm 0 0.6275

Calculated Concentrations 0 0.5289

Sample 3

0.435

0.510

0.4725

0.4324

Sample 4

0.298

0.368

0.333

0.4210

Sample 5

0.196

0.241

0.2185

0.3683

Sample 6

0.105

0.133

0.119

0.3009

Sample 7

0.048

0.089

0.0685

0.3464

Figure 1. Average Absorbance vs. BSA Concentration (mg/mL)

Standard Curvefor BovineSerumAlbumin Absorbanceat 280 nm 1.6 1.4

AbsorbanceValue

1.2 1 0.8

y =1.582x R² =0.9853

0.6 0.4 0.2 0 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

BSA Concentration (mg/mL) Figure 1. A standard curve is produced by plotting absorbance values at 280 nm against the concentrations of the standard (Bovine serum albumin). A linear equation is generated, y = 1.582x, where y and x represented absorbance value at 280 nm and BSA concentration in mg/mL respectively. The slope of the standard curve distinguishes the molar absorptivity constant at 280 nm to be 1.582 M-1cm-1.

In Table 1, the absorbance values of 7 samples at various BSA concentrations were taken. The volume of the bovine serum albumin (BSA) standard and phosphate-buffered saline (PBS) solution for each sample at specific BSA concentrations was provided in accompanying columns. I collected an absorbance value of 0.973 in trial one, at a BSA concentration of 1.5 mg/mL, and this was done for a total of 7 samples of BSA protein. The absorbance values of these 7 samples were taken in 2 trials and averaged to attain the average absorbance at 280 nm for each sample. This Table 2 displays the absorbance values discerned from each sample with the unknown

protein and PBS solution. For each of the 7 samples, 2 trials were conducted and the absorbance values collected at each trial was averaged to gather an average absorbance value at 280 nm for each sample. The final column of Table 2 displays the calculated concentrations, which are calculated using Beer’s law (A =   c  l) and multiple calculations using the C1V1=C2V2 equation (since a serial dilution scheme was utilized). Figure 1 displays a graph generated from the average absorbance values collected at different concentrations of BSA protein in Table 1. The y axis of this graph is the absorbance value and the x axis is the BSA concentration in mg/mL. Within this figure, the graph generated a R2 value to be 0.9853. This proves there is strong correlation between the extrapolated line of best fit and the data points, leading to the conclusion that the BSA protein followed Beer’s Law. The graph also has a line of best fit going through the data points. The line of best fit gives a linear equation of y = 1.582x, where y and x correlate to the horizontal and vertical axes of the graph. The slope from this line of best fit equates to the molar absorptivity constant (280) at 280 nm due to the assumptions that can be made from Beer’s Law. Beer’s Law states the concentration of a chemical is directly proportional to the absorbance of a solution. The equation for this law is Absorbance (A) =  (molar absorptivity constant) x l (path length of light) x c (concentration of the solution). Interpretation: The theoretical stock concentration of the unknown protein was expected to be 1.5 mg/mL. Our experimental data led us to obtain an average calculated stock concentration of unknown protein 2 to be 0.3996 mg/mL. The variation between the theoretical and experimental concentration values generated a percent error of 73.36%. Looking at figure 1, we are able to distinguish the R2 value to be 0.9853 and this proves the data for the BSA standard follows

Beer’s Law. The strong correlation between the extrapolated line of best fit and the data points leads to the conclusion that the BSA protein followed the expected results and the percent error is generated from the unknown protein values. The linear nature of the line in figure 1 also leads to the assumption that the error is not due to any possible errors with the spectrophotometer or errors due to calibration of the instruments. A feasible reason for 73.36% error is inaccuracy in creating the dilution samples for the protein standards. Since our experiment required a serial dilution to be conducted, many samples were exposed to the heat for longer periods of time than the earlier samples. The heat would affect the structure of the protein and impact the amount of light that would be refracted from it when the Ultraviolet spectroscopy was conducted. Another possible reason for error was that many samples were used repetitively for the serial dilution scheme and these samples could have been exposed to particles in the air that could negatively affect the light absorbance in the spectrophotometer. The serial dilutions also required multiple cuvettes to be used and there was a high possibility of mixing up cuvettes during the trial. An inaccuracy in protein concentration in a sample can have an adverse effect on the data because the absorbance value will not correlate with the trend. Overall, the major reason for our large percent error in the experiment is due to the fact that a serial dilution scheme was used. Explanation: The data in Table 1 shows that as the concentration of the BSA standard increases, the absorbance value increases directly. This positive correlation can be witnessed on Figure 1, since the data of Table 1 has been graphed on this image. The data points within Figure 1 show a positive correlation and a direct trend in the data. The line of best fit calculated the linear equation of the data to be y = 1.582x. Within this equation, the slope of the standard curve distinguishes the 280 to be 1.582 mL/mgcm. Table 2 shows similar data for the unknown

protein experiment. As the concentration of unknown protein in a sample increases, the correlating absorbance values increase. This trend within the data, for both the standard and unknown protein samples, is expected because as there are more molecules within the solution, more light gets blocked, and a larger absorbance value is detected. This molar absorptivity constant is specific at this wavelength of light (280 nm) and can be used within Beer’s law to determine the stock concentration of the unknown protein. By taking the absorbance of my unknown protein at different concentrations, I was able to use Beer’s law and the molar absorptivity constant I found previously to determine the concentration of that specific sample. That concentration will then be plugged into the C1V1=C2V2 to find the concentration of the subsequent sample. This scheme would continue until the stock concentration of the protein from each sample was found. Afterwards, we averaged all of the stock concentration values of the unknown protein to attain an average calculated stock concentration of unknown protein 2 to be 0.3996 mg/mL. Within this experiment, the negative control is the blank cuvette (sample 1 in both data tables) of only PBS buffer. This is labeled as the negative control because there were no expected results from it. The positive controls were the protein solutions because they provided results with varying absorbance values. If this experiment were to be conducted again, I would come into lab with the dilution scheme for quantification adjusted for a serial dilution and the volumes calculated to fit into a 1.2 mL cuvette. These adjustments would save time during lab, allow the allocated resources to be used efficiently, and would help avoid any confusion when preparing the samples. In our upcoming experiment, we will be performing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in order to determine the molecular weight of

our unknown protein. SDS-PAGE denatures proteins and gives them a negative uniform charge which allows the proteins to be separated based on size alone, with the smaller polypeptide chain migrating further than the larger polypeptide chains. The band migration lengths will help with the identification of the unknown protein. Overall, this upcoming protein characterization experiment will determine the molecular weight (in Daltons) of the unknown protein and this information will be utilized to narrow the unknown protein to a possible 2-3 known proteins. Future characterization experiments will then distinguish the identity of the unknown protein from the 2-3 possible known proteins....