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Autophagy LC3 HiBiT Reporter Assay System

LC3 HiBiT Reporter Autophagy Assay Detection Kit
LC3 HiBiT Reporter Autophagy Assays
Autophagy Assays Kit LC3 HiBiT Reporter
LC3 HiBiT Reporter Autophagy Assay Detection Kit
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Multimodal Assay for Detection of Autophagic Flux

  • Quantitative, unambiguous, scalable LC3 reporter assay
  • No-wash, add-mix-measure protocol
  • Blotting and imaging formats for confirmatory readings

Choose a reporter cell line or reporter vector

Catalog number selected: GA1040

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Autophagy LC3 HiBiT Reporter Assay System
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Simple Detection of Autophagic Flux and Autophagosome Formation

The Autophagy LC3 HiBiT Reporter Assay System uses a luminescent LC3 reporter to quantitatively measure autophagic flux. Choose from one of two prepared reporter cell lines (HEK293 or U2OS) or stably transfect the reporter vector into the cell line of your choice. With one assay, you can verify results using three different methods: plate reader, protein blotting and imaging.

How to Measure Autophagy Using the LC3 Assay

The Autophagy LC3 HiBiT Reporter consists of a HiBiT tag fused to the human LC3 protein, connected by a HaloTag® spacer. When autophagy is induced, LC3 reporter proteins are captured in autophagosomes and degraded. With addition of the Nano-Glo® HiBiT Lytic Reagent containing LgBiT protein and substrate, LgBiT interacts with the HiBiT tag to reconstitute the bright, luminescent NanoBiT® enzyme. The luminescent signal is directly proportional to the level of HiBiT-tagged LC3 reporter over seven orders of magnitude. Additional detection modules are HiBiT blotting and imaging with HaloTag® ligands.

how to measure autophagy lc3 hibit reporter assay

1 Assay for 3 Different Methods: Plate Reader, Protein Blotting and Imaging

autophagy lc3 hibit reporter assay formats

Plate Reader Data: Quantify Autophagy Inducer/Inhibitor Effect

With just a plate reader, you can easily quantify autophagy levels by detecting luminescence readout. Increased autophagic flux will accelerate the degradation of the autophagy reporter, resulting in decreased luminescent signal. In contrast, inhibition of autophagy will increase reporter levels and luminescent signal.

Autophagy Induction: Decreased Signal

Autophagy Assay Kit & Decreased Signal Data
HEK293 Autophagy LC3 HiBiT Reporter cells were plated into all-white, 96-well plates at 20,000 cells/well. After cells attached overnight, treatment was performed in triplicate for the indicated times with increasing concentrations of a reference autophagy inducer, PP242. Nano-Glo® HiBiT Lytic Reagent was then added and luminescence measured after 10 minutes. Average signal is reported as normalized luminescence relative to time-matched, vehicle-treated controls.

Autophagy Inhibition: Increased Signal

Autophagy Assay Kit & Increased Signal Data
U2OS Autophagy LC3 HiBiT Reporter Cells were plated into all-white, 96-well plates at 8,000 cells/well.  After cells attached overnight, cells were treated with increasing concentrations of a reference inhibitor, Baf A1, without or with 2μM PP242 for 21 hours. Luminescent signal was normalized to untreated control for each study. Treatment with PP242 significantly increased autophagy and decreased luminescent signal to allow more capacity for signal increase by cotreatment with the autophagy inhibitor, Baf A1.

See More Data: Learn more about this data and see how the Autophagy LC3 HiBiT Reporter Assay can be used with 3D cell models in the poster Assessing Autophagic Flux in 2D and 3D Cell Culture Models with a Novel Plate-Based Assay, presented at ASCB 2017. 

Download the Poster

Protein Blotting Data: Measure LC3-I to LC3-II Conversion

The HiBiT blotting method can detect HiBiT-tagged proteins on nitrocellulose membranes in as little as 5 minutes—without using antibodies! Luminescent signal is produced only where HiBiT is present, minimizing background. This method can discern LC3-I from lipidated LC3-II, and quantify proteins down to sub-picogram levels.
measure lc3i to lc3ii conversion protein blotting autophagosome
U2OS Autophagy LC3 HiBiT Reporter Cells were treated with PP242 (autophagy stimulator) alone or with BafA1 (late-phase pathway inhibitor) for 4 hours. PP242 alone stimulates autophagy and increases degradation of the reporter. With the addition of Baf A1, LC3-I is converted to LC3-II but the degradation of LC3-II is blocked and both LC3-I and LC3-II levels increase.

Imaging Data: Visualize LC3 Localization to the Autophagosome

Fluorescent imaging allows direct observation of autophagosome formation. The rapid binding kinetics of the HaloTag® spacer with HaloTag® ligands helps you visualize protein trafficking and turnover in the autophagosome. Visualization of LC3 reporter location from diffuse to autophagosome localization can help elucidate changes in the autophagy pathway.
how to measure autophagy autophagosome imaging
U20S Autophagy LC3 HiBiT Reporter Cells were treated for 4 hours with PP242, an autophagy stimulator, with WN or Baf A1, autophagy inhibitors. The localization of the LC3 reporter was imaged using Janelia Fluor® 646 HaloTag® Ligand. Images were acquired at 60X magnification. The stimulator (PP242) elevates autophagosome levels, which can be visualized as increased puncta compared to vehicle control. Early-phase pathway inhibitors (WN) prevent autophagosome formation (reduced puncta), while late-phase pathway inhibitors (Baf A1) elevate autophagosome formation (increased puncta).

Protocols

Complete Protocol

Quick Protocols

Specifications

You are viewing: GA1040 Change Configuration

What's in the box?

Item Part # Size Available Separately

Nano-Glo® HiBiT Lytic Detection System

 N3030 1 × 10ml View Product

HEK293 Autophagy LC3 HiBiT Reporter Cell Line

 GA104A 1 × 1 each

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Patents and Disclaimers

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If the researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.

This product may not be further sold or transferred by the researcher and may be used only by the researcher, and then only for research use, which includes, but is not limited to drug discovery and development. No other commercial use is allowed. "Commercial use" means any and all uses of this product by researcher for monetary or other consideration, including, but not limited to, (1) use in further product manufacture; (2) use in provision of services, information or data to unaffiliated third parties, and (3) resale of this product for any use. Researcher has no right to modify, derivatize, genetically engineer or otherwise create variations of the nucleotide sequence encoding the HiBiT and HaloTag® peptides which sequences have been stably transfected within the cells. Researcher may genetically engineer the cell line using exogenous nucleic acid sequences of the researcher’s choosing and researcher may propagate and store the cells for long-term use. In addition, researcher must use Nano-Glo® HiBiT Lytic Detection System purchased from Promega for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega's. For uses of HaloTag® Technology in this product, researchers must either: (1) use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties, or (2) contact Promega to obtain a license if Promega HaloTag® ligands are not to be used.

With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.

Researcher may use this product for research use only; no commercial use is allowed. Commercial use means any and all uses of this product by a party in exchange for consideration, including, but not limited to (1) use in further product manufacture; (2) use in provision of services, information or data; and (3) resale of the product, whether or not such product is resold for use in research. Researcher shall have no right to modify or otherwise create variations of the product. No other use or transfer of this product is authorized without the prior express written consent of Promega.

For uses of Nano-Glo®-branded reagents intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researchers must:
(i) use NanoBRET®-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product; or
(ii) contact Promega to obtain a license for use of the product for energy transfer assays to energy acceptors not manufactured by Promega.

With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. No. 9,797,890 and other patents pending.

Licensed from Kazusa Genome Technologies.

HEK293 cells were obtained under license from AdVec Inc.

Specifications

You are viewing: GA1050 Change Configuration

What's in the box?

Item Part # Size Available Separately

U2OS Autophagy LC3 HiBiT Reporter Cell Line

 GA105A 1 × 1 each

Nano-Glo® HiBiT Lytic Detection System

 N3030 1 × 10ml View Product

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Patents and Disclaimers

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If the researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.

This product may not be further sold or transferred by the researcher and may be used only by the researcher, and then only for research use, which includes, but is not limited to drug discovery and development. No other commercial use is allowed. "Commercial use" means any and all uses of this product by researcher for monetary or other consideration, including, but not limited to, (1) use in further product manufacture; (2) use in provision of services, information or data to unaffiliated third parties, and (3) resale of this product for any use. Researcher has no right to modify, derivatize, genetically engineer or otherwise create variations of the nucleotide sequence encoding the HiBiT and HaloTag® peptides which sequences have been stably transfected within the cells. Researcher may genetically engineer the cell line using exogenous nucleic acid sequences of the researcher’s choosing and researcher may propagate and store the cells for long-term use. In addition, researcher must use Nano-Glo® HiBiT Lytic Detection System purchased from Promega for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega's. For uses of HaloTag® Technology in this product, researchers must either: (1) use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties, or (2) contact Promega to obtain a license if Promega HaloTag® ligands are not to be used.

With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.

Researcher may use this product for research use only; no commercial use is allowed. Commercial use means any and all uses of this product by a party in exchange for consideration, including, but not limited to (1) use in further product manufacture; (2) use in provision of services, information or data; and (3) resale of the product, whether or not such product is resold for use in research. Researcher shall have no right to modify or otherwise create variations of the product. No other use or transfer of this product is authorized without the prior express written consent of Promega.

For uses of Nano-Glo®-branded reagents intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researchers must:
(i) use NanoBRET®-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product; or
(ii) contact Promega to obtain a license for use of the product for energy transfer assays to energy acceptors not manufactured by Promega.

With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. No. 9,797,890 and other patents pending.

Licensed from Kazusa Genome Technologies.

Product cannot be used for proficiency testing.

Specifications

You are viewing: GA2550 Change Configuration

What's in the box?

Item Part # Size Available Separately

Autophagy LC3 HiBiT Reporter Vector

 GA255A 1 × 20μg

Nano-Glo® HiBiT Lytic Detection System

 N3030 1 × 10ml View Product

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Patents and Disclaimers

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.

No transfer of this product is allowed. Researchers may use this product in their own research, which includes, but is not limited to drug discovery and development, and they may transfer derivatives to others for research use provided that at the time of transfer a copy of this label license is given to recipients and recipients agree to be bound by the terms of this label license. No other commercial use is allowed. "Commercial use" means any and all uses of this product by researcher for monetary or other consideration, including but not limited to, (1) use in further product manufacture; (2) use in provision of services, information or data to unaffiliated third parties, and (3) resale of this product for any use.

Researchers shall have no right to modify or otherwise create variations of the nucleotide sequences that encode the HiBiT and HaloTag® peptides. No other use of this product or its derivatives is authorized without the prior express written consent of Promega.

In addition, researchers must either:
(1a) use Nano-Glo® HiBiT Lytic Detection System-branded luminescent assay reagents (LARs) for all determinations of luminescence activity of this product and its derivatives; or
(1b) contact Promega to obtain a license for use of the product and its derivatives with LARs not manufactured by Promega.

For uses of Nano-Glo®-branded LARs intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researchers must either:
(2a) use NanoBRET®-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product and its derivatives; or
(2b) contact Promega to obtain a license for use of the product and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega.

For uses of HaloTag® Technology in this product, researchers must either: (1) use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties, or (2) contact Promega to obtain a license if Promega HaloTag® ligands are not to be used.

With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.

Researcher may use this product for research use only; no commercial use is allowed. Commercial use means any and all uses of this product by a party in exchange for consideration, including, but not limited to (1) use in further product manufacture; (2) use in provision of services, information or data; and (3) resale of the product, whether or not such product is resold for use in research. Researcher shall have no right to modify or otherwise create variations of the product. No other use or transfer of this product is authorized without the prior express written consent of Promega.

For uses of Nano-Glo®-branded reagents intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researchers must:
(i) use NanoBRET®-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product; or
(ii) contact Promega to obtain a license for use of the product for energy transfer assays to energy acceptors not manufactured by Promega.

With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. No. 9,797,890 and other patents and patents pending.

U.S. Pat. Nos. 7,425,436, 7,935,803, 8,466,269, 8,742,086, 8,420,367, 8,748,148, 9,416,353, 9,593,316 and other patents and patents pending.

Licensed from Kazusa Genome Technologies.

Resources

Articles

Citations

  • Multimodal assessment of autophagy in mammalian cells with a novel, LC3-based tandem reporter.
    --
    2021 Methods Cell Biol.
  • See all citations

Posters

Other Resources

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