|
|
| Home > JCE Print > Journal of Chemical Education > Issues >
2002
>
December
> |
|
In the Laboratory
|
|
|
|
C-SNARF-1 as a Fluorescent Probe for pH Measurements in Living Cells: Two-Wavelength-Ratio Method versus Whole-Spectral-Resolution Method
|
A-C. Ribou, J. Vigo, and J-M. Salmon
Laboratory of Physico-Chemical Biology of Integrated Systems, University of Perpignan,
F-66860, Perpignan, France
|
|
December 2002
Vol. 79 No. 12
p. 1471
|
|
|
|
|
|
|
| Abstract |
|
Measuring intracellular concentrations of ions (H+, Ca2+, Mg2+) is one of the challenges of modern cell biology. Fluorescence techniques can be used for this purpose since they are noninvasive, specific, and in general sensitive enough to require only low concentrations of the probes. In this paper, we describe two methods to measure pH by fluorescence spectroscopy and a way to extend the measurement to living cells. We use the fluorescent probe, C-SNARF-1, which exhibits a spectral shift of 45 nm upon proton binding. The two-wavelength-ratio method is applied to the determination of an unknown pH in aqueous solution. The whole-spectrum-resolution method is used for the same solution for comparison and then applied on a prerecorded spectrum of a suspension of living cells. The methods described here have been adapted for laboratory classes of undergraduate students. The lab employs experimental fluorescence techniques and computational data analysis that account for the fluorescence of cellular flavoproteins. The pedagogic purpose of the experimental design is to introduce students to the concepts, equations, and validity of both methods. In addition, skills in record keeping and data analysis using Microsoft Excel Solver are developed.
|
| Supplement |
|
Detailed instructions for students, notes for the instructor (preparation of the solutions, Excel data sheet of Figure 3), and handout are available.
Note: The PDF file includes the content of the Excel data sheet appended to the end.
|
 Contents |
Folder JCE2002p1471W containing JCE2002p1471W.doc (Microsoft Word) and JCE2002p1471W.xls (Microsoft Excel)
|
| Download |
|
|
| More Information |
Citation |
Ribou, A-C.; Vigo, J.; Salmon, J-M. J. Chem. Educ. 2002 79 1471.
|
Keywords |
Acid-Base Chemistry; Analytical Chemistry; Biophysical Chemistry; Equilibrium; Fluorescence Spectrometry; Laboratory Instruction; Physical Chemistry
|
History |
Created:
Last Updated: |
October 29, 2002
March 16, 2005
|
|
|
|
|
|
| Home > JCE Print > Journal of Chemical Education > Issues >
2002
>
December
> Page
1471
|
|
|
| JCE HS CLIC |
|
Our Secondary School editors work hard to distill all the JCE materials to produce a fraction of particular interest to high school teachers. We call it CLIC.
|
| Contributions Welcome |
| JCE welcomes your submission |
| Advertisers |
| In recent years we have worked hard to better match our advertisers with our readers. When shopping for chemistry education materials, visit our advertisers' WWW sites first. |
| Be An Ambassador |
| Take JCE along on your outreach missions. Copies of the Journal, guest access to JCE Online, our publications catalog, and more are available for your participants. |
|
