天津大学研究生e-Learning平台

An enzyme-linked immunosorbent assay (ELISA) was used to examine the accessibility of GFP11 in the BsLS+GFP11 complex. Purified protein samples of either free GFP11 BsLS or BsLS+GFP11 complex were each added to individual wells in an ELISA plate. The concentration of the BsLS+GFP11 sample was chosen to give the same amount of GFP11 as in the free GFP11 sample. The concentration of the BsLS sample was chosen to give the same concentration of BsLS as in the sample of BsLS+GFP11. An additional sample was prepared in which both GFP11 and BsLS that had each been produced and purified in the absence of each other were mixed together in the same well to see if exogenous BsLS can influence the accessibility of GFP11. For the initial protein adsorbing step, all sample volumes were 50 µL. Proteins were adsorbed to the wells overnight at 4 °C. On the second day,pour off the sample from the well,then the wells were washed with 1% PBST buffer(50 mM sodium phosphate, 150 mM NaCl, pH 7.4,1% Tween 20) for 3 times,every time need 5 min. Then every well was blocked by incubating in 5% non-fat milk(0.5 g non-fat milk was dissolved in 10 mL PBS buffer) for 2 h at 37 °C, followed by additional washing with PBST buffer for 3 times.Every time need to turn upside down the plate and dry it in tissue. 50 µL polyclonal rabbit anti-GFP antibody as the primary antibody was added to the well and incubate at 37 °C for 1 h .Pour the primary antibody from the plate followed washing with PBST for 3 times and every time is 5 min.Next a monoclonal goat anti-rabbit antibody conjugated to horseradish peroxidase as the secondary antibodies was added to the plate. Antibody binding step were carried out at 37 °C for 1 h .After finishing the binding step,50 µL PBST fill the well to wash the plate for 3 times. The relative amount of bound secondary antibody (and thus of accessible GFP11) was determined by incubating each well with a 50 µL 1% solution of the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine for 10 min at room temperature. Add 0.1% H₂O₂ when use the substrate buffer.After quenching with 50 µL 1 M H₂SO₄ (50 µL) the OD₄₅₀ was then measured. Assays were carried out in triplicate for each protein.