Colloidal coomassie

Depletion of hemoglobin from erythrocyte cytosol was modified from a previously published study by Ringrose et al [15 (link)]. Ni-NTA Superflow Columns (Qiagen, Venlo, Netherlands) were equilibrated with 10 ml NPI-10 buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0). 2 mL erythrocyte cytoplasm was loaded onto the Ni-NTA columns with gravity flow. Cytosolic proteins were eluted six times with 1 mL NPI-15 buffer (50 mM NaH₂PO₄, 300 mM NaCl, 15 mM imidazole, pH 8.0). In cases where detectable hemoglobin eluted during the NPI-15 washes, the red eluate was subjected to one more round of Hb-depletion in new equilibrated Ni-NTA columns and washed two times with 1 mL NPI-15. The Hb-enriched fraction was collected with four elutes of 1 mL NPI-250 buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0). The fraction containing the Hb-depleted cytosolic proteins was concentrated to approximately 2 mg/mL using 3 kDa molecular weight cutoff protein concentrators (Thermo Scientific, Rockford, IL). The final protein concentration was determined using a Modified Lowry Protein Assay kit (Thermo Scientific). Samples were analyzed by SDS-PAGE on 12-well 10% Bis-Tris gels (Invitrogen, Carlsbad, CA) followed by staining with colloidal Coomassie (Invitrogen).

For recombinant protein production, Escherichia coli BL21 (DE3) cells (Novagen) grown overnight at 37 °C in 25 ml LB broth containing 100 μg/ml ampicillin, and 25 μg/ml kanamycin were inoculated into 500 ml LB broth, and grown at 37 °C. At OD600_nm of 0.8, IPTG was added to concentration of 0.75 mM, and the culture was further incubated for 3 h at 30 °C. Cells were pelleted, resuspended in 25 ml lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4)), and suspended cells were lysed by micro-fluidiser. The cell lysates were then spun at 15,500 rpm for 20 min, at 4 °C. The cleared supernate, containing both GST-tagged recombinant proteins, was removed and subsequently purified by standard Glutathione Sepharose purification method. Purified proteins were fractionated on a Novex NuPAGE 4–12% bis-Tris gel (Invitrogen) and stained with colloidal Coomassie (Roth).