green fluorescent protein gfp tagged variant  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: The Membrane Proximal Domain of TRPV1 and TRPV2 Channels Mediates Protein–Protein Interactions and Lipid Binding In Vitro

doi: 10.3390/ijms20030682

Figure Lengend Snippet: Mapping of the TRPV interaction with SNARE proteins. ( a ) Co-IP of human embryonic kidney 293 (HEK293) cells transfected with TRPV2 lacking N-terminal ARD domain GFP-tagged (ΔARD-TRPV2-GFP) and Snapin-25 or Synaptotagmin-IX c-myc-tagged. The lysates were immunoprecipitated with anti-c-myc. ( b ) In vitro pull-down assay using purified MPD region from TRPV1 or TRPV2 channels GFP-tagged to pull-down c-myc-tagged Snapin or Synaptotagmin-IX in lysates from HEK293 transiently transfected cells. MPDs-GFP were immobilized on GFP-Trap beads. IP: immunoprecipitation; IB: immunoblotting. Co-IP experiments have been carried out in the absence (−) and presence (+) of c-myc antibody to assess the specificity of the interaction.

Article Snippet: Primary antibodies were diluted as follows: anti-MYC tag (551101, Pharmingen, Germany) 1:1000, anti-GFP tag (GFP-G1, DSHB, Iowa, IA, USA) 1:1000.

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, In Vitro, Pull Down Assay, Purification, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Membrane Proximal Domain of TRPV1 and TRPV2 Channels Mediates Protein–Protein Interactions and Lipid Binding In Vitro

doi: 10.3390/ijms20030682

Figure Lengend Snippet: Interaction of MPD domain from TRPV1 and TRPV2 with phosphatidic acid (PA). ( a ) Blank (only buffer), PI, PA, PI4P, PS, PC, or PG lipids were immobilized on nitrocellulose membranes at concentrations (conc.) of 50, 100, or 200 μM. Purified recombinant GFP or MPD-GFP from TRPV1 or TRPV2 channels were incubated on the lipid-containing nitrocellulose membranes and detected using an anti-GFP antibody. ( b ) Tryptophan quenching experiments. GFP tryptophan fluorescence (emission at 333 nm) of free GFP or MPDs from TRPV1 or TRPV2 was monitored in the presence of increasing concentrations of the different lipid polar head-groups (Phosphatidylcholine, POPC, 1,2-Dipalmitoyl-sn-glycero-3-phosphate (DPPA), and Phosphatidylglycerol (POPG)) liposomes.

Article Snippet: Primary antibodies were diluted as follows: anti-MYC tag (551101, Pharmingen, Germany) 1:1000, anti-GFP tag (GFP-G1, DSHB, Iowa, IA, USA) 1:1000.

Techniques: Purification, Recombinant, Incubation, Fluorescence