ip extraction buffer  (Thermo Fisher)

Journal: bioRxiv

Article Title: UPL3 Promotes BZR1 Degradation, Growth Arrest, and Seedling Survival under Starvation Stress in Arabidopsis

doi: 10.1101/2023.10.18.562997

Figure Lengend Snippet: A) Workflow of SILIP-MS experiments for identifying BZR1-interacting proteins in sugar-starved Arabidopsis plants. Two-week seedlings grown on 14 N- or 15 N-labeled 1/2MS medium without sugar were treated by darkness for 24 hrs, then were harvested for immunoprecipitation. In two forward-labeling replicate experiments (F1 and F2), the 35S:BZR1-YFP and 35S:GFP plants were labeled with 14 N and 15 N, respectively, whereas the isotopes were switched in the reverse labeling replicate (R1). B) Western blots show the BZR1-YFP or GFP protein immunoprecipitated by GFP-trap. C) Coomassie blue-stained SDS-PAGE gels of proteins immunoprecipitated by GFP-trap from 14 N/ 15 N-labelled BZR1-YFP or GFP . Each lane was cut into 5 pieces as shown in the image for mass spectrometry analysis.

Article Snippet: After grinding with liquid nitrogen, the tissues were resuspended in immunoprecipitation (IP) extraction buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10% glycerol, 0.25% Triton X-100, 0.25% NP40,1 mM PMSF, Pierce protease inhibitor cocktail [Thermo Fisher Scientific], and PhosStop cocktail [Roche]), and sonicated for 20 seconds (1s on/off).

Techniques: Labeling, Immunoprecipitation, Western Blot, Staining, SDS Page, Mass Spectrometry

Journal: bioRxiv

Article Title: UPL3 Promotes BZR1 Degradation, Growth Arrest, and Seedling Survival under Starvation Stress in Arabidopsis

doi: 10.1101/2023.10.18.562997

Figure Lengend Snippet: A-C) PRM quantitation of 14 N- and 15 N-labeled peptides of UPL3 and ACC1, as ratios between BZR1-YFP and GFP control, in three replicates of anti-GFP immunoprecipitation. D) Co-IP assay shows in vivo interaction between BZR1-YFP and UPL3-Myc. N. benthamiana leaves were transiently co-transformed with BZR1:BZR1-YFP (or 35S:GFP as a control) and 35S:UPL3-Myc. The Input extract and anti-GFP immunoprecipitants (IP) were analyzed by immunoblotting (IB).

Article Snippet: After grinding with liquid nitrogen, the tissues were resuspended in immunoprecipitation (IP) extraction buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10% glycerol, 0.25% Triton X-100, 0.25% NP40,1 mM PMSF, Pierce protease inhibitor cocktail [Thermo Fisher Scientific], and PhosStop cocktail [Roche]), and sonicated for 20 seconds (1s on/off).

Techniques: Quantitation Assay, Labeling, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, In Vivo, Transformation Assay, Western Blot