Difference between revisions of "Part:BBa K5097017:Experience"

Revision as of 19:01, 28 September 2024

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Applications of BBa_K5097017

E. coli DH5 alpha cells expressing this construct were lysed using lysozyme and sonication, His tagged RFP was purified by Ni-IMAC chromatography and eluted in 50mM sodium phosphate, 300mM sodium chloride, and 500mM imidazole at pH8.0. After concentration and buffer exchange into PBS, the purity of the protein product was visualized after SDS-PAGE and coomassie staining. (Figure 1). Experiments were performed to understand the impact pH would have on the fluorescence of their reporter system.

Figure 1: Coomassie-stained PAGE gel showing purification of BFP-6XHis and RFP-6XHis.

RFP was loaded into a 96-well plate and buffer was added to a final volume of 100 µL, yielding a protein concentration of 0.760 ng/ml. The buffers used range from pH 4.0 to 9.0. 100μL of each buffer alone was added in another row, and fluorescence was then measured using Fluorescence Spectroscopy. An emission scan quantified by signal intensity runs from 575 nm to 665 nm with a fixed excitation wavelength of 555nm.

Figure 2: Graph comparing the maximum intensity emitted at each pH tested

No significant shift was observed in the maximum emission wavelength*

User Reviews

UNIQ4e1ddcb810853d6e-partinfo-00000000-QINU UNIQ4e1ddcb810853d6e-partinfo-00000001-QINU

@@ Line 5: / Line 5: @@
 ===Applications of BBa_K5097017===
+E. coli DH5 alpha cells expressing this construct were lysed using lysozyme and sonication, His tagged RFP was purified by Ni-IMAC chromatography and eluted in 50mM sodium phosphate, 300mM sodium chloride, and 500mM imidazole at pH8.0. After concentration and buffer exchange into PBS, the purity of the protein product was visualized after SDS-PAGE and coomassie staining. (Figure 1).  Experiments were performed to understand the impact pH would have on the fluorescence of their reporter system.
+[[File:bba-k509013-figure1.webp|Figure 1: Coomassie-stained PAGE gel showing purification of BFP-6XHis and RFP-6XHis.]]
+RFP was loaded into a 96-well plate and buffer was added to a final volume of 100 µL, yielding a protein concentration of 0.760 ng/ml. The buffers used range from pH 4.0 to 9.0.  100μL of each buffer alone was added in another row, and fluorescence was then measured using Fluorescence Spectroscopy. An emission scan quantified by signal intensity runs from 575 nm to 665 nm with a fixed excitation wavelength of 555nm.
+[[File:bba-k5097017-figure2.webp|Figure 2: Graph comparing the maximum intensity emitted at each pH tested]]
+*No significant shift was observed in the maximum emission wavelength*
 ===User Reviews===