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Method for determining type of microtubule-associated protein-1 light chain 3 protein spot

A technology for microtubule-related proteins and spots, which is used in material excitation analysis, fluorescence/phosphorescence, etc. to improve accuracy

Active Publication Date: 2013-05-22
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, existing research results show that although FP-LC3G120A (FP-LC3□G) does mainly label protein aggregates and not autophagic structures under certain conditions, in some cases FP-LC3G120A (FP-LC3 □G) will still enter the autophagic vesicle, thereby marking the autophagic structure

Method used

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  • Method for determining type of microtubule-associated protein-1 light chain 3 protein spot
  • Method for determining type of microtubule-associated protein-1 light chain 3 protein spot
  • Method for determining type of microtubule-associated protein-1 light chain 3 protein spot

Examples

Experimental program
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Embodiment 1

[0037] In this example, the LC3 protein (dendra2-LC3) fused to dendra2 photoconversion protein was first transfected into HeLa cells for 24 hours. Cells were then treated with puromycin for 2.5 hours, and dendra2-LC3 spots were induced. Puromycin is a widely used reagent to induce autophagic puncta formation. Finally, the FRAPa method was used for analysis. figure 1 It is the figure of FRAPa technology to determine the type of dendra2-LC3 spots induced by puromcyin. It can be seen from the figure that after the dendra2-LC3-labeled protein aggregates are activated by light, the fluorescence at the spots decreases rapidly and significantly.

Embodiment 2

[0039] In this example, GFP-fused LC3 protein (GFP-LC3) and mCherry-fused LC3 protein (mCherry-LC3G120A) were first co-transfected into HeLa cells for 24 hours. The cells were then treated with puromycin (Rapamycin) and chloroquine (CQ for short) for 6 hours. Rapamycin and CQ are widely used reagents to induce autophagic puncta formation. Finally, the FRAP method was used to analyze, figure 2 is the negative untreated group and the control group, image 3 are the graphs of CQ-induced GFP-LC3 / mCherry-LC3G120A puncta, Figure 4 Figures for rapamycin-induced GFP-LC3 / mCherry-LC3G120A puncta, Figure 5 is a graph of the statistics of the average intracellular GFP-LC3 / mCherry-LC3G120A spots in the negative control group, Image 6 is a graph of the statistics of the average intracellular GFP-LC3 / mCherry-LC3G120A spots in the CQ-treated group, Figure 7is a graph of the statistics of the average intracellular GFP-LC3 / mCherry-LC3G120A spots in the rapamycin-treated group, Figur...

Embodiment 3

[0041] In this example, GFP-LC3 and mCherry-LC3G120A were first co-transfected into HeLa cells for 24 hours. Cells were then treated with LY294002, wortmannin and MG132 for 6 hours. LY294002, wortmannin and MG132 are reported chemicals that induce protein aggregates. Finally, the FRAP method was used to analyze, Figure 10 Figures for LY294002-induced GFP-LC3 / mCherry-LC3G120A puncta, Figure 11 Figures for wortmannin-induced GFP-LC3 / mCherry-LC3G120A puncta, Figure 12 Figures for MG-132-induced GFP-LC3 / mCherry-LC3G120A puncta, Figure 13 Detection of LY294002-induced GFP-LC3 for FRAP technology + / mCherry-LC3G120A + Graph of the kinetics of spotted GFP-LC3; Figure 14 Detection of wortmannin-induced GFP-LC3 by FRAP technique + / mCherry-LC3G120A + Graph of the kinetics of spotted GFP-LC3; Figure 15 Detection of MG-132-induced GFP-LC3 for FRAP + / mCherry-LC3G120A + Graph of the kinetics of spotted GFP-LC3. from Figures 10 to 15 As can be seen, a certain amount of ...

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Abstract

The invention provides a method for determining the type of a microtubule-associated protein-1 light chain 3 protein spot and belongs to the technical field of cell imaging. The method comprises the following steps of: observing whether a complete and rapid exchange behavior exists between the microtubule-associated protein-1 light chain 3 protein (FP-LC3) spot link-coupled with a fluorescent protein and FP-LC3 in cell plasma of a mammal; if so, determining that the FP-LC3 spot is in the type of a protein aggregate; and if few or no exchange behavior exists between the FP-LC3 and the FP-LC3 in the cell plasma of the mammal, determining that the FP-LC3 spot is in the type of an autophagy structure. By using the method, the type of the LC3 can be accurately and rapidly determined.

Description

technical field [0001] The invention belongs to the technical field of cell imaging, and in particular relates to a method for determining the type of microtubule-associated protein 1 light chain 3 protein spot. Background technique [0002] In the field of autophagy research, it is often necessary to characterize intracellular autophagic structures. Microtubule-associated protein 1 light chain 3 (LC3) protein is considered to be a marker protein for autophagy. Fluorescent (FP) protein and LC3 are usually fused and expressed in mammalian cells, so that the formation and dynamic changes of autophagic structures can be dynamically observed by fluorescence microscopy. In fluorescence microscopy imaging, fluorescent protein-microtubule-associated protein 1 light chain 3 (FP-LC3) molecules are uniformly distributed in the cytoplasm and nucleus or speckled structures in the cytoplasm, the latter mainly representing Autophagy structure. [0003] However, the latest research resu...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 骆清铭张智红王亮
Owner HUAZHONG UNIV OF SCI & TECH
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