Difference between revisions of "Part:BBa K3133004"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | BBa_K3133004 mTagRFP-mWasabi-LC3 | + | ==BBa_K3133004 mTagRFP-mWasabi-LC3== |
<p>Method:</p> | <p>Method:</p> | ||
<p>Activation on autophagy was investigated by using mTagRFP-mWassbi-LC3 translocation assay. Briefly, SH-SY5Y cells were transfected with mRFP-GFP-LC3 plasmid via Lipofectamine 2000 according to the manufacturer’s protocol. The cells were treated with different testing compounds and then fixed with 4% paraformaldehyde. The images were acquired using a Leica DMI8 confocal microscope and analyzed by the Image J software. Autophagy activator Nitazoxanide (NTZ) and inhibitor Chloroquine (CQ) were applied to an assay of SH-SY5Y as positive and negative control, respectively.</p> | <p>Activation on autophagy was investigated by using mTagRFP-mWassbi-LC3 translocation assay. Briefly, SH-SY5Y cells were transfected with mRFP-GFP-LC3 plasmid via Lipofectamine 2000 according to the manufacturer’s protocol. The cells were treated with different testing compounds and then fixed with 4% paraformaldehyde. The images were acquired using a Leica DMI8 confocal microscope and analyzed by the Image J software. Autophagy activator Nitazoxanide (NTZ) and inhibitor Chloroquine (CQ) were applied to an assay of SH-SY5Y as positive and negative control, respectively.</p> | ||
Revision as of 12:28, 18 October 2019
mTagRFP-mWasabi-LC3
mWasabi-LC3 was used because it was more acid sensitive than EGFP-LC3 in lysosomes and brighter when monitoring autolysosomes in autophagy flux. Also, the exposure time needed by mWasabi-LC3 is shorter than the one of EGFP-LC3, which yielded photos of higher quality. Because LC3 could also be present in non-autophagy cells, mWasabi-LC3∆G is more effective at discriminating them during autophagy flux (Zhou 2012).
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 622
Illegal BsaI.rc site found at 760
Illegal BsaI.rc site found at 1171