Enzymatic assay kit

The recombinant EPO secreted into the cell culture medium was puri ed by reversed-phase chromatography (Yoon et al. 2005) (link). EPO was separated from culture supernatant by loading the samples onto a reversed-phase column (RESOURCE RPC 3 mL, GE Healthcare). The EPO was separated from the samples by utilizing gradient elution: eluent A was composed of 10 mM Tris (pH 7), and eluent B was composed of 10 mM Tris and 80% ethanol (pH 7). The peak related to EPO was observed at a concentration of 65% ethanol, and SDS-PAGE and Western blot assessed the purity of obtained EPO. Then, the buffer of puri ed EPO protein was exchanged with deionized water through the dialysis process at 4 o C.
Determination of glucose, ammonia, lactate, and EPO sialic acid contents
The glucose content of samples was determined by an enzymatic assay kit (Abcam, USA), as per kit instructions. Ammonia concentration was measured based on an ammonia detection kit (Sigma-Aldrich, USA) regarding the instruction manual. According to kit instructions, lactate determination was conducted based on a lactate assay kit (R-Biopharm, Germany). The sialic acid content of puri ed EPO was determined by utilizing the EnzyChrom sialic acid assay kit (BioAssay Systems, CA), following the manufacturer's protocol.

The required amount of CHO cells (depending on the test) was collected and were incubated in cold methanol for 5 min. Then, collected samples were washed twice, resuspended in cold PBS, and stored at -80 o C for further analysis. CHO cells were lysed via sonication and were centrifuged at 10000 g for 10 min (4 o C). The supernatant was used for the measurement of α-ketoglutarate concentration and citrate synthase activity. The concentration of α-ketoglutarate was measured by a microplate assay kit (Abcam, USA), following the kit's instructions. Citrate synthase was quanti ed with an enzymatic assay kit (Abcam, USA), as per kit instructions.