<p> <h3>Characterization of chromoproteins</h3> </p>
<p> <h3>Characterization of chromoproteins</h3> </p>
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Different types of body fluid have different pH (figure). Since we are going to use body fluid as sample in our influenza diagnostic test, we would like to investigate if the pH in body fluid can interfere with the reporter protein we used in our test. Fluorescent signal is known to be pH-dependent because pH can change the folding and conformation of the fluorophore, and ionization states can also cause shift in the Excitation/Emission spectra (Ref). Therefore, we characterized the fluorescence of 2 fluorescent proteins: mRFP and amajLime at different pH. We want to find out their optimum pH and see if they are suitable to be the reporter protein in our diagnostic test.
Different types of body fluid have different pH (figure). Since we are going to use body fluid as sample in our influenza diagnostic test, we would like to investigate if the pH in body fluid can interfere with the reporter protein we used in our test. Fluorescent signal is known to be pH-dependent because pH can change the folding and conformation of the fluorophore, and ionization states can also cause shift in the Excitation/Emission spectra (Ref). Therefore, we characterized the fluorescence of 2 fluorescent proteins: mRFP and amajLime at different pH. We want to find out their optimum pH and see if they are suitable to be the reporter protein in our diagnostic test.
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To examine the performance of mRFP and amajLime under different pH, we inserted the biobricks into pSB1A2 and expressed them in <i>E. coli</i>. mRFP and amajLime were then purified form lysed cells by anion exchange chromatography (AEC) and hydrophobic interaction chromatography (HIC).
To examine the performance of mRFP and amajLime under different pH, we inserted the biobricks into pSB1A2 and expressed them in <i>E. coli</i>. mRFP and amajLime were then purified form lysed cells by anion exchange chromatography (AEC) and hydrophobic interaction chromatography (HIC).
F.1 to F.3 represents chronological order of elutions in HIC. Samples (volume?) were mixed with 10 µl 2X SDS gel-loading buffer and 10 µl of the mixture were loaded on the SDS-gel. The purest fraction, F.2 in both cases, were selected to proceed to pH stability test, where purified proteins were diluted in buffers in the range of pH 2-12 to a final concentration of ZZZ mg/mL, and the fluorescence intensity at XXX nm (Ex YYY nm) was recorded by BMG ClarioStar plate reader.
F.1 to F.3 represents chronological order of elutions in HIC. Samples (volume?) were mixed with 10 µl 2X SDS gel-loading buffer and 10 µl of the mixture were loaded on the SDS-gel. The purest fraction, F.2 in both cases, were selected to proceed to pH stability test, where purified proteins were diluted in buffers in the range of pH 2-12 to a final concentration of ZZZ mg/mL, and the fluorescence intensity at XXX nm (Ex YYY nm) was recorded by BMG ClarioStar plate reader.