β lactoglobulin b

The proteins used throughout this work were trypsin (Sigma, catalog No. T1426), β-lactoglobulin B (Sigma, catalog No. L8006) and concanavalin A (ConA). ConA was purified in-house from jack beans using the method of Agrawal & Goldstein (1967 ▸ ). The purified ConA was stored in crystalline form at 4°C. An aliquot of the precipitate was removed, concentrated by centrifugation, dissolved in weak ammonium hydroxide solution and underwent buffer exchange using a centrifugal desalting column into the crystallization buffer 0.025 M Na HEPES, 0.025 M NaCl pH 7.5.

The following specimens were purchased from Sigma-Aldrich (St. Louis, MO, USA): (1) human serum albumin (fatty acid and human globulin free (~99%)), (2) albumin from bovine serum, (3) human γ-globulin, (4) β-lactoglobulin A from bovine milk (>90%), (5) β-lactoglobulin B from bovine milk (>90%), (6) lysozyme from chicken egg white, (7) trypsinogen from bovine pancreas, (8) ovalbumin, and (9) pyridine-N-oxide (PyO) with a purity of 95% for the NMR measurements and analysis.

Di-sodium hydrogen phosphate (Na₂HPO₄), sodium dihydrogenphosphate dihydrate (NaH₂PO₄·2H₂O), sodium chloride (NaCl), L-lysine hydrochloride (C₆H₁₄N₂O₂·HCl), tetrachloroauric acid (HAuCl₄), sulfuric acid (H₂SO₄), and potassium chloride (KCl) were acquired from Merck (Milan, Italy). potassium hexacyanoferrate (II) trihydrate (K₄[Fe(CN)₆]·3H₂O), potassium hexacyanoferrate (III) (K₃[Fe(CN)₆]), Trizma^® Base (Tris), magnesium chloride hexahydrate (MgCl₂·6H₂O), β-lactoglobulin B (β-LG), and 6-mercapto-1-hexanol (MCH) were obtained from Sigma-Aldrich (Milan, Italy).
The thiolated aptamer used, modified in the 3’-end with methylene blue (MB), had the following sequence: 5’-HS-CGA CGA TCG GAC CGC AGT ACC CAC CCA CCA GCC CCA ACA TCA TGC CCA TCC GTG TGT G-MB-3’, and was obtained from Biomers.net (Ulm, Germany).
All the reagents used were of analytical grade and used as they were received, without the need of further purification. Milli-Q water (18 MΩ) was used to prepare all the solutions.