Pika Hemoglobin Isoform Analysis

We used isoelectric focusing (PhastSystem, GE Healthcare Bio-Sciences, Piscataway, NJ) to characterize the Hb isoform composition of circulating red blood cells in both O. princeps and O. collaris. Native pika Hbs were purified by means of anion-exchange chromatography (HiTrap QHP, GE Healthcare, Piscataway, NJ), as described previously (Storz et al. 2012 (link); Revsbech et al. 2013 (link)). The column was pre-equilibrated with 20 mM Tris (pH 8.4) and the sample was eluted using a linear gradient of 0–0.2 M NaCl. Samples were desalted by dialysis against 10 mM HEPES buffer (pH 7.4) and were concentrated using Millipore centrifugal filter units (30-kDa cutoff). Hb concentrations were determined from absorbance spectra using standard extinction coefficients at 577 and 540 nm.

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Tufts D.M., Natarajan C., Revsbech I.G., Projecto-Garcia J., Hoffmann F.G., Weber R.E., Fago A., Moriyama H, & Storz J.F. (2014). Epistasis Constrains Mutational Pathways of Hemoglobin Adaptation in High-Altitude Pikas. Molecular Biology and Evolution, 32(2), 287-298.

Publication 2014

PhastSystem HiTrap QHP

Anion Buffer Chromatography Dialysis Extinction Hepes Isoform Nacl Pika Red blood cells Tris

Corresponding Organization : University of Nebraska–Lincoln

Other organizations : Aarhus University, Mississippi State University, Biocom

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Variable analysis

independent variables

Isoelectric focusing (PhastSystem, GE Healthcare Bio-Sciences, Piscataway, NJ) used to characterize the Hb isoform composition of circulating red blood cells
Anion-exchange chromatography (HiTrap QHP, GE Healthcare, Piscataway, NJ) used to purify native pika Hbs

dependent variables

Hb isoform composition of circulating red blood cells in both O. princeps and O. collaris
Purified native pika Hbs

control variables

Column pre-equilibrated with 20 mM Tris (pH 8.4)
Samples eluted using a linear gradient of 0–0.2 M NaCl
Samples desalted by dialysis against 10 mM HEPES buffer (pH 7.4)
Samples concentrated using Millipore centrifugal filter units (30-kDa cutoff)
Hb concentrations determined from absorbance spectra using standard extinction coefficients at 577 and 540 nm

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

Annotations

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