Quantitative Analysis of Globin Chains in Erythroblasts

Analysis of globin chains in CD34⁺-cell-derived erythroblasts was performed by reverse-phase HPLC, as previously described^{48 (link),49 (link)}. In brief, the reverse-phase HPLC assay was carried out on an Agilent 1260 Infinity II HPLC system with Diode-Array Detector. The chromatographic column used was an Aeri 3.6-μm WIDEPORE XB-C18 200-Å, LC Column 250 × 4.6 mm² behind a securityGuard ULTRA cartridge (Phenomenex). Globin chains were separated using a gradient program of 41–47% solvent B (acetonitrile) mixed with solvent A (0.1% trifluoroacetic acid in HPLC-grade water at pH 2.9), and quantified by the area under the curve (AUC) of the corresponding peaks in the reverse-phase HPLC chromatogram.

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Cromer M.K., Camarena J., Martin R.M., Lesch B.J., Vakulskas C.A., Bode N.M., Kurgan G., Collingwood M.A., Rettig G.R., Behlke M.A., Lemgart V.T., Zhang Y., Goyal A., Zhao F., Ponce E., Srifa W., Bak R.O., Uchida N., Majeti R., Sheehan V.A., Tisdale J.F., Dever D.P, & Porteus M.H. (2021). Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells. Nature medicine, 27(4), 677-687.

Publication 2021

1260 Infinity II HPLC system securityGuard ULTRA cartridge

Acetonitrile Assay Cell Chromatographic Erythroblasts Globin Hplc Solvent Trifluoroacetic acid

Corresponding Organization :

Other organizations : Stanford University, Integrated DNA Technologies (United States), Baylor College of Medicine, Aarhus University, National Heart Lung and Blood Institute, National Institutes of Health, California Institute for Regenerative Medicine

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Variable analysis

independent variables

Gradient program of 41–47% solvent B (acetonitrile) mixed with solvent A (0.1% trifluoroacetic acid in HPLC-grade water at pH 2.9)

dependent variables

Globin chain composition as quantified by the area under the curve (AUC) of the corresponding peaks in the reverse-phase HPLC chromatogram

control variables

Agilent 1260 Infinity II HPLC system with Diode-Array Detector
Aeri 3.6-μm WIDEPORE XB-C18 200-Å, LC Column 250 × 4.6 mm^2 behind a securityGuard ULTRA cartridge (Phenomenex)
PH 2.9 of the HPLC-grade water used in solvent A

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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