Difference between revisions of "Part:BBa K5301009"
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<p>We also employed AlphaFold to predict the structure of the protein constituted by SpyCatcher, mCherry[1-10], spNW15, mCherry[11] and SpyTag, and discovered that following the successful conjugation of SpyTag and SpyCatcher, mCherry[1-10] and mCherry[11] were successfully complemented.(Figure 1). </p> | <p>We also employed AlphaFold to predict the structure of the protein constituted by SpyCatcher, mCherry[1-10], spNW15, mCherry[11] and SpyTag, and discovered that following the successful conjugation of SpyTag and SpyCatcher, mCherry[1-10] and mCherry[11] were successfully complemented.(Figure 1). </p> | ||
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We used Western Blot to test whether the protein containing mCherry[11] had been expressed successfully(Figure 2). The molecular weight of SnTST- mCh[11] (containing mCherry[11]) is 33.6 kDa. And we purified the target protein with a molecular weight of approximately 25-34 kDa (The red box), which demonstrated that we had successfully expressed the protein containing mCherry[11]. | We used Western Blot to test whether the protein containing mCherry[11] had been expressed successfully(Figure 2). The molecular weight of SnTST- mCh[11] (containing mCherry[11]) is 33.6 kDa. And we purified the target protein with a molecular weight of approximately 25-34 kDa (The red box), which demonstrated that we had successfully expressed the protein containing mCherry[11]. | ||
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Furthermore, we employed a Fluorescent Inverted microscope to examine whether mCherry[1-10] successfully complemented mCherry[11] and emitted fluorescence (Figure 3). We observed the red fluorescence of mCherry under the Fluorescent Inverted microscope, demonstrating that they functioned successfully. | Furthermore, we employed a Fluorescent Inverted microscope to examine whether mCherry[1-10] successfully complemented mCherry[11] and emitted fluorescence (Figure 3). We observed the red fluorescence of mCherry under the Fluorescent Inverted microscope, demonstrating that they functioned successfully. | ||
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| + | Figure 3.The mCherry fluorescence observation chart (10×10) under green light excitation. It was observed using a fluorescent Inverted microscope and photographed with an ordinary mobile phone. | ||
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| + | ===Sequence and Features=== | ||
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Latest revision as of 11:49, 1 October 2024
MCherry [11] is a part of bimolecular fluorescence complementation (BiFC) system.
MCherry [11] is a part of bimolecular fluorescence complementation (BiFC) system which could visualize protein interactions. The combination of mCherry [1-10] and mCherry [11] can form the fluorescence of mCherry. With its advantages of a short maturation time and brilliant fluorescence, the mCherry BiFC system could find particular applications for analyzing protein–protein interactions especially in living cells [1].
Usage and Biology
We devised the fusion expression of mCherry[11] with spNW15, SpyTag and SnoopCatcher (namely SnTST- mCh[11]). We used mCherry[1-10] and mCherry[11] parts to emit fluorescence to verify the success of protien connection. If SCSdC-mCh[1-10] can connect to SnTST- mCh[11] successfully, the mCherry[1-10] and mCherry[11] parts can emit fluorescence as a characterization.
We also employed AlphaFold to predict the structure of the protein constituted by SpyCatcher, mCherry[1-10], spNW15, mCherry[11] and SpyTag, and discovered that following the successful conjugation of SpyTag and SpyCatcher, mCherry[1-10] and mCherry[11] were successfully complemented.(Figure 1).
Figure 1.The structure of the protein constituted by SpyCatcher, mCherry[1-10], spNW15, mCherry[11] and SpyTag as predicted by AlphaFold
Characterization
We used Western Blot to test whether the protein containing mCherry[11] had been expressed successfully(Figure 2). The molecular weight of SnTST- mCh[11] (containing mCherry[11]) is 33.6 kDa. And we purified the target protein with a molecular weight of approximately 25-34 kDa (The red box), which demonstrated that we had successfully expressed the protein containing mCherry[11].
Figure 2.Western Blot analysis of the extraction results of SnTST- mCh[11](containing mCherry[11]).The molecular weight of SnTST- mCh[11] is 33.6kDa.
Furthermore, we employed a Fluorescent Inverted microscope to examine whether mCherry[1-10] successfully complemented mCherry[11] and emitted fluorescence (Figure 3). We observed the red fluorescence of mCherry under the Fluorescent Inverted microscope, demonstrating that they functioned successfully.
Figure 3.The mCherry fluorescence observation chart (10×10) under green light excitation. It was observed using a fluorescent Inverted microscope and photographed with an ordinary mobile phone.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
- ↑ Fan, J., et al., Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein–protein interactions in living cells. Biochemical and Biophysical Research Communications, 2008. 367(1): p. 47-53.