Instrument system gold 126 solvent module 166 detector

Analysis of HbA, HbF and free α-globin chains was performed with HPLC as elsewhere reported [24 (link),58 (link),61 (link),63 (link)]. Lysates have been loaded into a PolyCAT-A cation exchange column and then eluted in a sodium-chloride-BisTris-KCN aqueous mobile phase using HPLC Beckman Coulter instrument System Gold 126 Solvent Module-166 detector, which allows to obtain for the quantification of the hemoglobins present in the sample. Further details can be found in Supplementary Materials (SM7 and SM9).

K562 cells were harvested, washed once with PBS and the pellets were lysed in lysis buffer (sodium dodecyl sulphate 0.01%). After incubation on ice for 15 min, and spinning for 5 min at 14000 rpm in a microcentrifuge, the supernatant was collected and injected. Hb proteins present in the lysates were separated by cation-exchange HPLC [25 (link), 35 (link)], using a Beckman Coulter instrument System Gold 126 Solvent Module-166 Detector. Hemoglobins were separated using a PolyLC (Columbia, MD, USA) PolyCAT-A model (35 mmx4.6 mm) column; samples were eluted in a solvent gradient using aqueous sodium chloride-BisTris-KCN buffers and detection was performed at 415 nm. The standard controls were the purified HbA (SIGMA, St Louis, MO, USA) and HbF (Alpha Wassermann, Milano, Italy).