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Detecting protein unfolding with a UV light pen
Author(s) -
West Lori,
Sherwood Laura,
Bergeron Sean,
DeLaLuz Paul
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.444.10
Subject(s) - green fluorescent protein , fluorescence , urea , folding (dsp implementation) , chemistry , protein folding , biophysics , lysis , biochemistry , chromatography , biology , optics , gene , physics , electrical engineering , engineering
Green fluorescent protein (GFP) has become increasingly useful in both research and educational settings because of its ease of detection. Our goal was to develop a low tech way in which to use GFP to demonstrate the effects of denaturants on protein folding. This technique would be especially practical for high schools and small colleges where access to specialized equipment is limited. Crude lysate from E. coli cells expressing GFP was used to monitor the effects of SDS and urea on fluorescence and hence folding of GFP. We first confirmed that GFP fluorescence could be observed visually by excitation with a UV light pen (Bio‐Rad®). We then denatured the protein with 5M urea or 1% SDS and again monitored fluorescence. We were unable to detect fluorescence as expected for denatured GFP. We confirmed these results by performing an unfolding curve with concentrations of urea between 1M and 5M and SDS between 0.25% and 1%. Although we were unable to visually detect a significant change in fluorescence at the intermediate concentrations of urea and SDS, we were able to observe differences between fully folded GFP and unfolded GFP using a UV light pen.