Bovine pancreatic insulin

1-Anilino-8-naphthalene sulfonic acid (ANS), Thioflavin T (ThT), α-chymotrypsin, kanamycin, isopropyl-1-thio-β-d-galactopyranoside (IPTG), α-glucosidase (α-Gls), dithiothreitol (DTT), 2,5-diphenyltetrazolium bromide (MTT) and bovine pancreatic insulin were purchased from Sigma. Additionally, we used ethylenediaminetetraacetic acid (EDTA), urea, β-mercaptoethanol (β-ME), and various other chemicals, which were provided by Merck.

MCF7, MDAMB231, T47D, MCF10A, Hs578T and HEK293 cell lines were obtained from ATCC and grown according to their guidelines. MDAMB361 cells (ATCC) were grown in DMEM (Gibco Invitrogen) with 20% fetal bovine serum (FBS; Gibco Invitrogen) and 1% antibiotics (Gibco Invitrogen). B80T5 cells (a gift from Roger Reddel; CMRI, Australia) were grown in RPMI (Gibco Invitrogen) with 10% FBS and 1% antibiotics. K5+/K19 + cells [12 (link)] were grown in 1:1 MEM α (Gibco Invitrogen) and Ham’s F-12 Nutrient Mix (Gibco Invitrogen) with 1% FBS, 10mM HEPES, 1 µg/ml bovine pancreatic insulin, 1 µg/ml hydrocortisone, 50 µg/ml epidermal growth factor (Sigma Aldrich), 10 mg/ml transferrin, 100 µM β-estradiol, 2 mM glutamine, 2.6 ng/ml sodium selenite, 1 ng/ml cholera toxin (Sigma Aldrich), 6.5 ng/ml triiodothyronine, 100 µM ethanolamine, 35 µg/ml BPE, 10 µg/ml gentamicin, 10 µg/ml ascorbic acid, 15 µg/ml hygromycin B. All cell lines were tested for mycoplasma contamination and verified by short tandem repeats (STR) profiling.

The chemicals hen egg white lysozyme (>0.95), bovine pancreatic insulin (>0.95), L-proline (>0.99), sorbitol (>0.95) and thioflavin T (dye content 0.65–0.75) were procured from Sigma-Aldrich Chemical Company USA. The listed purities of these compounds, on mass fraction basis, are given in the parenthesis. All the solutions were prepared in milliQ water from Merk Millipore system. The lysozyme solution was prepared in 40 mM phosphate buffer at pH 7.0 and insulin solution was prepared in 20 mM phosphate buffer at pH 2.0. The stock solutions of each protein were dissolved initially in the respective buffers and dialyzed overnight extensively at 4°C against the buffer with at least three changes of the latter. All other solutions were prepared in the final dialysate buffer. The pH of the solution was measured on a Pico+ pH meter from Lab India Pvt. Ltd. at ambient temperature.

Bovine pancreatic insulin (>0.95), L-proline (>0.99), citrulline (>0.95), trehalose (>0.95), sorbitol (>0.95), glycine betaine (>0.95) and thioflavin T (dye content 0.65–0.75) were procured from Sigma-Aldrich Chemical Company USA. The listed purities of these compounds, on mass fraction basis, are given in the parenthesis. All the solutions were prepared in milliQ water from Merk Millipore. The experiments were performed in 20 mM phosphate buffer at pH 2.0. The stock solution of insulin was prepared initially in the phosphate buffer (20 mM, pH 2.0) and dialyzed overnight at 4° C against the buffer with at least three changes of the latter. All the other solutions were prepared in the final dialysate buffer.