Biochemical and biophysical analysis of i-α1-ECD.

<p><b>A</b>) <b>Gel filtration analysis</b>. The right y axis shows the absorbance at 280 nm (A₂₈₀) and the left y axis the bound ¹²⁵I-α-BTX (Δcpm) in the filter assay, using 20 μL of each gel filtration fraction and 3,000 cpm of ¹²⁵I-α-BTX. The values are the mean and standard deviation (±S.D.). Arrows indicate the peaks of eluted protein markers of known molecular mass. <b>B</b>) <b>Far-UV CD analysis</b>. Deconvolution of the presented spectrum with CDPro software showed 8% α-helical content, 40% β-sheet structure, 22% β-turns and 30% unordered portion. NRMSD (normalized root mean square deviation) value was 0.05, reflecting the accurate analysis of secondary structure. <b>C</b>) <b>DLS analysis</b>. The size distribution by volume of the gel-filtration isolated i-α1-ECD molecules is shown. The calculated hydrodynamic diameter of i-α1-ECD is 4 nm with a polydispersity value of 29%. Protein sample was derived from gel filtration analysis and its concentration was 0.3 mg/mL. SDS-PAGE analysis followed by D) Coomassie Brilliant Blue staining and <b>E</b>) Western blot analysis using the anti-α1 nAChR mAb 198. Lane 1: Positions of protein Mr standards; Lane 2: y-α1-ECD; Lane 3: i-α1-ECD. Protein samples were derived from gel filtration chromatography.