Journal: BMC Plant Biology

Article Title: Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes

doi: 10.1186/s12870-015-0660-9

Figure Lengend Snippet: Wheat germ-based in vitro ubiquitination analysis of RING proteins showed high molecular smears. a At4g11680 and its corresponding RING mutant were expressed with biotin tag and analysed as a representative protein in the presence or absence of FLAG-Ub, E1, and/or AtUBC8 without tag for the ubiquitination activity. Replacement of the third metal ligand, Cys to Ser, caused reduced smear upon blotting against FLAG-Ub with anti-FLAG-HRP antibody. Absence of the E1 or AtUBC8 from the reaction did not abolish the high molecular smear. b Three other RING proteins At1g80400, At2g22120, and At1g11020, and their RING mutants together with At4g11680, were analysed in the presence of FLAG-Ub. The four proteins with RING mutants showed significantly reduced activity upon blotting with anti-FLAG-HRP antibody, whereas the proteins with intact RING domains showed activity without the addition of E1 or E2. The side arrow refers to the free FLAG-Ub that migrated to the bottom of the gel

Article Snippet: In a total reaction volume of 25 μL, we mixed 20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 3 mM ATP, 1 mg/ml BSA, 400 nM human recombinant FLAG-Ub (Boston Biochem, http://www.bostonbiochem.com ), 3 μL recombinant E2s.

Techniques: In Vitro, Mutagenesis, Activity Assay

Journal: BMC Plant Biology

Article Title: Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes

doi: 10.1186/s12870-015-0660-9

Figure Lengend Snippet: Wheat germ-based in vitro ubiquitination assays of various types of RING proteins. 23 FLAG-tagged RING proteins of various RING types were mixed with FLAG-Ub in the presence or absence of N-bio-UBC10 as indicated by plus (+) or minus (–) above each lane. E3 activity was determined by the presence or absence of a smear when FLAG-Ub was detected by anti-FLAG-HRP antibody. This is indicated by plus (+) or minus (–), respectively, below each lane respectively

Article Snippet: In a total reaction volume of 25 μL, we mixed 20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 3 mM ATP, 1 mg/ml BSA, 400 nM human recombinant FLAG-Ub (Boston Biochem, http://www.bostonbiochem.com ), 3 μL recombinant E2s.

Techniques: In Vitro, Activity Assay

Journal: BMC Plant Biology

Article Title: Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes

doi: 10.1186/s12870-015-0660-9

Figure Lengend Snippet: Thioester assay of 35 Arabidopsis E2s. The crude proteins for each of the 35 bio-E2s were incubated with FLAG-Ub for 5 min at 37 °C and treated with DTT or 8 M urea (-DTT). Immunoblot analysis against FLAG-Ub using anti-FLAG-HRP antibodies shows the presence of DTT-sensitive Ub conjugation activity for all E2s tested. Arrows show the expected E2-Ub adduct for each E2 in the absence of DTT. The side arrows show the free FLAG-Ub and expected FLAG-Ub adducts with WE2 and WE1 as arranged from bottom to top

Article Snippet: In a total reaction volume of 25 μL, we mixed 20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 3 mM ATP, 1 mg/ml BSA, 400 nM human recombinant FLAG-Ub (Boston Biochem, http://www.bostonbiochem.com ), 3 μL recombinant E2s.

Techniques: Incubation, Western Blot, Conjugation Assay, Activity Assay

Journal: BMC Plant Biology

Article Title: Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes

doi: 10.1186/s12870-015-0660-9

Figure Lengend Snippet: Immunoblot analysis of the N-terminal FLAG-tagged RING protein library expressed by the wheat germ cell-free system. For analysis, 2 μL of crude recombinant RING proteins with N-terminus FLAG tag was loaded onto SDS-PAGE and detected by anti-FLAG-HRP antibody. A total of 204 out of 208 RING proteins analysed were detected. Arrows show the expected signal for each RING protein. Blue asterisks refer to proteins with high molecular smears, while red asterisks refer to RING proteins did not show high molecular smears and were subsequently used in the in vitro ubiquitination analysis (Fig. , Fig. )

Article Snippet: In a total reaction volume of 25 μL, we mixed 20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 3 mM ATP, 1 mg/ml BSA, 400 nM human recombinant FLAG-Ub (Boston Biochem, http://www.bostonbiochem.com ), 3 μL recombinant E2s.

Techniques: Western Blot, Recombinant, FLAG-tag, SDS Page, In Vitro