KIT LC3 trap-F for Western-blot

Product Description

LC3 traps were developed to isolate endogenous LC3/GABARAP proteins from mammalian cells (1). LC3 traps were engineered to contain multimeric LC3-interacting regions (LIRs) to increase their affinity for LC3/GABARAP proteins in various cell lines. LC3 traps F are based in 8 consensual LIR sequences with predominant hydrophobic amino-acids F typically found in the autophagy receptor OPTN. These LIRs are known to display affinity and specificity towards one or several LC3/GABARAP family members. LC3 traps F show binding preference towards GABARAP, GABARAP-L1 and GABARAP-L2 proteins in mammalian cells, and for LGG1 in C. elegans model. The affinity of LC3-traps was measured using microscale thermophoresis (MST) and estimated in the low nanomolar range (1).

B Molecular proposes LC3 trap F tagged with GST that can be attached to glutathione agarose affinity columns. LC3-traps can be easily use in GST-pull down assays to capture LC3/GABARAP proteins and cellular factors directly or indirectly associated to these ubiquitin family members from mammalian cells but also equivalent molecules LGG1 in C. elegans or ATG8 in S. cerevisiae models (1, 2). 

KIT LC3 Trap-F contains all reagents and solutions required for the capture of GABARAP, GABARAP-L1 and GABARAP-L2 proteins from cell lines, and distinct biologic models.  

 

 Applications 

-Capture of GABARAP, GABARAP-L1 and GABARAP-L2 proteins as well as complexes associated to these proteins from cell lines and model organisms followed by Western blot detection. 

-Immunoprecipitation of complexes associated to GABARAP, GABARAP-L1 and GABARAP-L2 proteins. This application requires the elution of captured proteins, dialysis and immunoprecipitation with a specific antibody recognizing a protein of interest that are not provided in this kit.  

Specificities 

LC3-traps F      are not crosslinked to agarose beads but preserved in a lyophilized state for long term storage. LC3-traps F      are reconstituted with a specific buffer just before use and mixed with glutathione agarose beads provided in the kit. Washing and elution buffers are also included. Negative control GST is provided for a single control reaction.

Captured proteins have to be boiled to be analyzed by Western blot. Since LC3-traps are not crosslinked to agarose, these will be released during the boiling. Avoid using antibodies for Western blot analysis that have been generated with a GST-protein of interest fusion, otherwise GST protein will be detected. 

Characteristics

  • Tag: GST 
  • Purity: > 95% by RP-HPLC and SDS-PAGE
  • Molecular Weight: 41,54 kDa
  • Physical State: Solid (powder)
  • Quantity: 250 µg      
  • Solubility: <5 mg/mL
  • Concentration: Variable
  • Storage: RT but once reconstituted store at -80°C and avoid freeze/thaw cycles

Benefits

  • Higher affinity than the single LIR domain
  • Avoid overexpression of tagged ATG8 proteins for pull downs
  • Replace LC3/GABARAP specific antibodies for enrichment of these proteins
  •     Kit LC3 Trap-F has been developed to facilitate the use, long-term stability and transport of all components required to capture proteins associated to LC3/GABARAP using agarose beads. 

Examples of use

Kit LC3 Trap-F      can be used to detect LC3/GABARAP proteins and cellular factors directly or indirectly associated to them implicated in the regulation of autophagy and signaling pathways regulating inflammation, oncogenesis, neurodegenerative diseases and multiple infections (3). The lipidated  status of LC3/GABARAP proteins can be followed in response to a treatment and determine if cells are responding well (1, 2). 

Publications

1. Quinet G, Génin P, Ozturk O, Belgareh-Touzé N, Courtot L, Legouis R, Weil R, Cohen MM, Rodriguez MS (2021) Exploring selective autophagy events in multiple biologic models using LC3-Interacting Regions (LIR)-based molecular traps. Scientific Reports. In press. 

2. Quinet G, Ozturk O, Belgareh-Touzé N, Legouis R, Cohen MM, Rodriguez MS (2021) Capture of ATG8 family members using LC3-traps. MiMB. In press. 

3. Jiang, P. & Mizushima, N. Autophagy and human diseases. Cell Res 24, 69–79 (2014).