User:Trisha I. Ibeh/Notebook/Trisha Notebook/2013/09/04

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Objective

Finish up the lab from the previous day (9/3/13) by taking the UV- Vis spectrum of inosine and doing data analysis. From the data, a Grubb's test will be performed to determine the outliers. The concentration of the unknown sample will be determined ad the molar absorbtivity will also be determined from the class data.

Procedure

The same procedure that was performed with adenosine will be performed with inosine. The procedure can be found here.

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




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