User:Andrew K Farag/Notebook/Andrewfarag/2013/09/04

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Objective

Become familiar with the fluorescence spectrometer and with protein fluorescence.

Description

Fluorescence spectroscopy is another way to analyze molecular samples.

Instructions

Each group will pick a different protein (bovine serum albumin, horseradish peroxidase, pepsin, adenosine deaminase, hemoglobin). You'll need to make 4 samples ranging from 10uM to 0.5uM. Remember, we don't have a lot of most of these proteins, so figure out how to make a stock solution using as little solid protein as possible. The fluorescence cuvette needs only 200uL, so you don't need to make more than that for each concentration that you're measuring.

  1. Make your solutions
  2. Take an absorbance measurement of your samples and a blank
  3. Take a fluorescence measurement of your samples and a blank (excitation: 290nm, emission: 310nm-500nm)
  4. Analyze your data. (Integrate your corrected spectrum. Integration is MUCH more important in fluorescence spectra than peak height.) Here's a site from the web that talks about how to integrate using formulas in excel.
  5. Normalize your corrected spectra to the absorption intensity at 280nm for the corresponding absorbance spectrum. (That is, divide the fluorescence intensity by the A(280) and plot all of the fluorescence spectra on the same chart. Do they match up? Are they different? What could this mean?)
    • Note --- We are only going to have 1 cuvette to use. I expect you to work quickly and spend time analyzing data.

Notes

I made a stock solution of BSA today. Here is the info:

BSA solution 0.0104g BSA (MW = 66776g/mol) in 0.0100mL water → 15.6μM

Data

UV-Vis Inosine Spectra is taking in lab, and the data of the calibration curve at 248 nm is shown in the graph below. Image:Absorptionvsconcentrationinosine.png
Inosine group calibration curve is shown in the graph below as well. Image:Inosinepooled.png

Calculations Using class Data

Adenosine's average absorbance is 0.250606061, and its standard deviation is 0.152263534.
The confidence level is calculated using the following equation:
 \left[ \bar{x} - \frac{ts}{\sqrt{n}}, \bar{x} + \frac{ts}{\sqrt{n}} \right] \,
At 90% confidence, the confidence interval is [0.204,0.296].
at 95% confidence, the confidence interval is [0.195,0.305].


Inosine's average absorbance is 0.299666667, and its standard deviation is 0.169381.
The confidence level is calculated using the following equation:
 \left[ \bar{x} - \frac{ts}{\sqrt{n}}, \bar{x} + \frac{ts}{\sqrt{n}} \right] \,
At 90% confidence, the confidence interval is [0.243,0.356].
at 95% confidence, the confidence interval is [0.231,0.368].


Grubb's test for outliers is used on the Adenosine data to determine if there are any outliers because there are a few data points that may appear to be outliers.


G = \frac{x_-\bar{x}}{s}


if Gcalc>Gtable, then the point is an outlier.
From the G-table, G=1.71.
From the data, points (2.00E-05,0.406),(2.50E-05,0.442), and (3.00E-05,0.558) seemed to be outliers.
The new graph for Adenosine is below.
Image:Adenosinegroupcorrected.png

Extrapolation-Unknown

The wavelength of the Inosine unknown is 249 with absorbance of 0.05. After correcting the best fit line, Absorbance=11007*Concentration.Accordingly, absorbance of 0.05 would result a concentration of 4.54*10-6M Actual concentration is 2.37-5M. The percent difference error on the calculated concentration is 422.0%




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