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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: 2015-03-18
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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Effect test

Embodiment 1

[0037] In this example, the LC3 protein (dendra2-LC3) fused with dendra2 phototransformation protein was first transfected into HeLa cells for 24 hours. Then the cells were treated with puromycin for 2.5 hours, and the formation of dendra2-LC3 spots was induced. Puromycin is a widely used reagent that induces autophagic speck formation. Finally, the FRAPa method was used for analysis. figure 1 It is a figure for determining the type of dendra2-LC3 spots induced by puromcyin by FRAPa technology. 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, first, GFP-fused LC3 protein (GFP-LC3) and mCherry-fused LC3 protein (mCherry-LC3G120A) were co-transfected into HeLa cells for 24 hours. Then the cells were treated with puromycin (Rapamycin) and chloroquine (chloroquine, abbreviated as CQ) for 6 hours. Rapamycin and CQ are widely used reagents to induce autophagy speck formation. Finally, the FRAP method was used for analysis. figure 2 It is the picture of negative untreated control group, image 3 is the graph of CQ-induced GFP-LC3 / mCherry-LC3G120A spots, Figure 4 is the graph of GFP-LC3 / mCherry-LC3G120A spots induced by rapamycin, Figure 5 It is a statistical graph of the average GFP-LC3 / mCherry-LC3G120A spots in the cells in the negative control group, Image 6 is a statistical graph of the average GFP-LC3 / mCherry-LC3G120A spots in cells in the CQ treatment group, Figure 7It is a statistical graph of the mean GFP-LC3 / mCherry-LC3G120A spots in cells in the rapamycin treatment group, Figur...

Embodiment 3

[0041] In this example, GFP-LC3 and mCherry-LC3G120A were firstly 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 the chemical reagents reported to induce protein aggregation. Finally, the FRAP method was used for analysis. Figure 10 is the graph of GFP-LC3 / mCherry-LC3G120A spots induced by LY294002, Figure 11 It is a graph of wortmannin-induced GFP-LC3 / mCherry-LC3G120A spots, Figure 12 MG-132 induces GFP-LC3 / mCherry-LC3G120A spots, Figure 13 Detection of GFP-LC3 induced by LY294002 for FRAP technique + / mCherry-LC3G120A + Diagram of the kinetics of spotted GFP-LC3; Figure 14 Detection of wortmannin-induced GFP-LC3 by FRAP technique + / mCherry-LC3G120A + Diagram of the kinetics of spotted GFP-LC3; Figure 15 Detection of MG-132-induced GFP-LC3 for FRAP technique + / mCherry-LC3G120A + Diagram of the kinetics of spotted GFP-LC3. from Figure 10 to Figure 15 ...

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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 spot type of microtubule-associated protein 1 light chain 3 protein. 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 as a marker protein of autophagy structure. Fluorescent (FP) protein and LC3 are usually fused and expressed in mammalian cells, so that the formation and dynamic changes of autophagy structures can be dynamically observed by fluorescence microscopy. In fluorescence microscopy imaging, fluorescent protein-microtubule-associated protein 1 light chain 3 protein (FP-LC3) molecules can be seen uniformly distributed in the cytoplasm and nucleus or a spot-like structure distributed in the cytoplasm, the latter mainly representing Autophagy structure. [0003...

Claims

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Application Information

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