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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> Biomaterials Design Lab</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Entry title==
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* Insert content here...
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==Objective==
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Become familiar with the fluorescence spectrometer and with protein fluorescence.
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==Description==
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Fluorescence spectroscopy is another way to analyze molecular samples.
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==Instructions==
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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.
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# Make your solutions
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# Take an absorbance measurement of your samples and a blank
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# Take a fluorescence measurement of your samples and a blank (excitation: 290nm, emission: 310nm-500nm)
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# Analyze your data. (Integrate your corrected spectrum. Integration is MUCH more important in fluorescence spectra than peak height.) [http://people.oregonstate.edu/~haggertr/487/integrate.htm Here's] a site from the web that talks about how to integrate using formulas in excel.
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# 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?)
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**Note --- We are only going to have 1 cuvette to use. I expect you to work quickly and spend time analyzing data.
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==Notes==
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I made a stock solution of BSA today. Here is the info:
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BSA solution 0.0104g BSA (MW = 66776g/mol) in 0.0100mL water → 15.6μM
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[[Category:Course]]
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[[Category:Miscellaneous]]
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[[Category:Course]]
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[[Category:Miscellaneous]]
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Revision as of 14:26, 4 September 2013

Biomaterials Design Lab Main project page
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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




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