FPLC was performed using an ÄKTA Explorer system. Reverse-phase FPLC was conducted on the n-butanol extract of C. sp. Panama (resuspended in start buffer) using a RESOURCE RPC 3 mL (GE Healthcare Life Sciences) column under gradient elution between 2% and 85% methanol in water 0.1% TFA at a constant 2 mL/min flow rate. Fraction F was collected, dried under vacuum, and the resulting residue resuspended in 20% DMSO. Size exclusion FPLC of this sample was performed on a Superdex Peptide 10/300 GL (GE Healthcare Life Sciences) column under isocratic elution with 20% DMSO in water at a constant flow rate of 1 mL/min.
Resource rpc 3 ml
Manufactured by GE Healthcare
The RESOURCE RPC 3 mL is a laboratory equipment product manufactured by GE Healthcare. It is a centrifuge tube designed to hold 3 milliliters of liquid sample. The product specification and technical details are factual and unbiased.
Automatically generated - may contain errors
2 protocols using resource rpc 3 ml
1
Purification of Bioactive Compounds
2
Purification and Analysis of Recombinant EPO
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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.
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.
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