Journal: Scientific Reports

Article Title: High resolution X-ray and NMR structural study of human T-cell immunoglobulin and mucin domain containing protein-3

doi: 10.1038/s41598-018-35754-0

Figure Lengend Snippet: Human TIM-3 IgV domain binding with 10 mM Ca ++ by NMR. ( a ) Plot of peak shifts showing 15 N-HSQC combined chemical shift changes index, expressed as [(ΔHcs/0.1 ppm) 2 + (ΔNcs/0.5 ppm) 2 ] 1/2 ,of hTIM-3 IgV upon binding with 10 mM Ca ++ . The data columns are colored according to degree of index changes (red > 1.0; green > 0.5; yellow > 0.25). The chemical shift change indices of Met118, Asn119 and Glu121 in F-G loop of hTIM-3 exceeded 2.0, even at well below the saturation concentration of Ca ++ binding. ( b ) Chemical shift changes of hTIM-3 backbone amides induced by 10 mM Ca ++ binding, mapped on to hTIM-3 crystal structure surface and colored by degree of index changes as in ( a ). The opposite BED and AGFCC′C″ faces are shown in the left and right panels, respectively, with the B-C, C-C′ and F-G loops marked in italics.

Article Snippet: For binding studies between human TIM-3 Fc fusion protein (BPS Bioscience) and tagless hCEACAM1 IgV domain, 50 μl of 250 nM tagless hCEACAM1 protein in Tris-buffered saline buffer containing 10 mM CaCl 2 (TBS-Ca ++ ) or TBS-Ca ++ buffer alone was added to wells of an ELISA plate and incubated overnight at 4 °C.

Techniques: Binding Assay, Concentration Assay

Journal: Scientific Reports

Article Title: High resolution X-ray and NMR structural study of human T-cell immunoglobulin and mucin domain containing protein-3

doi: 10.1038/s41598-018-35754-0

Figure Lengend Snippet: Human TIM-3 and human CEACAM1 binding studies by ELISA. ( a ) hTIM-3 Fc fusion protein but not hIgG-Fc fusion protein (0–4 μM) binds to tagless hCEACAM1 IgV domain with EC 50 of 1.50 μM. ( b ) Blockade of the interaction between tagless hCEACAM1 and hTIM-3 Fc fusion protein with 10 μM of hTIM-3 C-C′ loop-derived peptide (amino acids 58–77) but not by 10 μM of scrambled peptide. ( c ) Binding of tagless hTIM-3 IgV domain with glutathione-S-transferase (GST) tagged hCEACAM1 IgV-domain protein (0–15 μM) with EC 50 of 3.13 μM, but not with GST protein (0–15 μM). ( d ) Significant blockade of interaction between tagless hTIM-3 IgV domain and glutathione-S-transferase (GST) tagged hCEACAM1 IgV-domain fusion protein by a mouse anti-human CEACAM1 IgV-domain specific monoclonal 5F4 antibody (0–1 μM), but not by a isotype control MOPC antibody. ( a–d ) ELISA binding assays were performed in triplicate and the average values are shown with standard deviations. n.s., not significant. p values ≤ *0.05, **0.01, and ***0.001.

Article Snippet: For binding studies between human TIM-3 Fc fusion protein (BPS Bioscience) and tagless hCEACAM1 IgV domain, 50 μl of 250 nM tagless hCEACAM1 protein in Tris-buffered saline buffer containing 10 mM CaCl 2 (TBS-Ca ++ ) or TBS-Ca ++ buffer alone was added to wells of an ELISA plate and incubated overnight at 4 °C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay