Journal: Neuroscience

Article Title: Identification of Neuroglobin-interacting Proteins Using Yeast Two-hybrid Screening

doi: 10.1016/j.neuroscience.2011.10.046

Figure Lengend Snippet: Expression of Ngb-Gal4 BD fusion protein in yeast. Yeast strain Y187 was transformed with pGBKT7-Ngb and transformants were selected on appropriate SD medium. Soluble protein extracts were prepared and protein samples were subjected to SDS-PAGE. Ngb expression was detected using anti-Ngb antibody. Lane 1: control Y187 transformed with pGBKT7 vector. Lane 2: Y187 transformed with pGBKT7-Ngb.

Article Snippet: Mouse Ngb cDNA was amplified by PCR; the PCR fragment was then digested with Nde I and Bam HI, and inserted into the pGBKT7 vector (Clontech) to generate a construct of mouse Ngb cDNA fused in-frame to the GAL4 DNA-binding domain (BD) (amino acids [a.a.] 1–147 of GAL4) as the bait.

Techniques: Expressing, Transformation Assay, SDS Page, Plasmid Preparation

Journal: Neuroscience

Article Title: Identification of Neuroglobin-interacting Proteins Using Yeast Two-hybrid Screening

doi: 10.1016/j.neuroscience.2011.10.046

Figure Lengend Snippet: Selection of Ngb-interacting gene products in yeast two-hybrid screening. Mating reaction was set up between Y187 transformed with pGBKT7-Ngb vector and AH109 pretransformed with mouse cDNA library. Representative selected clones were shown. (A) Positive clones in both Drop Out medium and alpha-glactosidase activity test. (B) Clones positive in Drop Out medium selection but negative in alpha-glactosidase activity.

Article Snippet: Mouse Ngb cDNA was amplified by PCR; the PCR fragment was then digested with Nde I and Bam HI, and inserted into the pGBKT7 vector (Clontech) to generate a construct of mouse Ngb cDNA fused in-frame to the GAL4 DNA-binding domain (BD) (amino acids [a.a.] 1–147 of GAL4) as the bait.

Techniques: Selection, Two Hybrid Screening, Transformation Assay, Plasmid Preparation, cDNA Library Assay, Clone Assay, Activity Assay