Journal: International journal of biological macromolecules

Article Title: Disulfide bond and crosslinking analyses reveal inter-domain interactions that contribute to the rigidity of placental malaria VAR2CSA structure and formation of CSA binding channel.

doi: 10.1016/j.ijbiomac.2022.11.258

Figure Lengend Snippet: Fig. 8. The binding capacity of full-length VAR2CSA ectodomain is lower than the ectodomain lacking NTS. The binding of NTS-DBL6ε and DBL1x-DBL6ε to CSPG was measured in triplicates by ELISA. The wells of 96-well microtiter plates were coated with CSPG (200 ng/ml) and blocked with BSA and then incubated with 1:2 serially diluted solutions of NTS-DBL6ε or DBL1x-DBL6ε. The levels of bound proteins were measured using anti-cMyc antibody and HRP- conjugated secondary antibody. Data are a representative of three independent experiments using different batches of purified proteins. KD values were determined by plotting OD vs protein concentration using GraphPad Prism v9.4.1. KD ± SE: 41.5 ± 3.9 (DBBL1x-DBL6ε); 59.9 ± 6.8 (NTS-DBL6ε).

Article Snippet: Unbound proteins in the coated wells were removed by washing with 100 μl of PBST and the plates were incubated with 1:1000 diluted mouse anti-cMyc monoclonal antibody (Cat No. NB600-302, NOVUS Biologicals, Centennial, CO), and washed three times with 100 μl PBST.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Purification, Protein Concentration