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Applications : Pull-down assay
Review: Competitive binding assays of CgRuby1 and CgRuby2Short binding to CgbHLH1. The mixture of HIS-CgRuby1 and FLAG CgRuby2Short was added to immobilized GSTCgbHLH1. The precipitates were detected using western blot analysis with anti-HIS, anti-FLAG or anti-GST antibodies. The gradient indicates the increasing amount of FLAG-CgRuby2Short. These experiments were repeated independently twice with similar results.
Applications : ELISA
Sample dilution: 1: 4000
Review: Murine B16F10 and L929 cell lines (2× 10 5 ) were fixed on plates and then blocked with 2% BSA solution. After that, Rechistatin, Echistatin, LgRec1 or EGFP (1; 3; 6 and 9μM) were added to the plates followed by incubation for 2 h at 37°C. The binding of the recombinant proteins to the cells was assessed using anti-His (Cusabio®) mAb followed by anti-mouse IgG-peroxidase conjugate (Sigma® ). The reaction was measured by spectrophotometer at 492 nm, the higher the absorbance the grater the binding of the toxins to the cells. A) Binding of recombinant toxins on murine B16F10 melanoma cells; B) Binding of recombinant toxins on murine L929 cells. The results were expressed as mean ± SD (n = 3), ***p < 0.001; **p < 0.01 and *p < 0.05 comparing Rechistatin, Echistatin, LgRec1 and EGFP groups com- pared with PBS.
Applications : GST pull-down
Sample dilution: 1:200
Review: We examined Pit-1 protein binding in relation to ESRRG alternative splicing status. Pit-1 protein fused to glutathione S-transferase (GST) pull-down HIS-tagged cryptic ESRRG, whereas it bound weakly to canonical ESRRG