Lysates from epimastigotes (2 × 109 parasites grown in glass Erlenmeyer flasks with mild agitation) were fractionated using a high-density gradient of iodixanol to isolate acidocalcisomes, as previously described (42 (link)). The fraction containing acidocalcisomes was resuspended in intracellular buffer, and 0.1 ml was resuspended in 3-ml quartz cuvettes for acridine orange incorporation measurements, as detailed below. Additions were made as detailed in figure legends. To analyze the content of the obtained fractions, we performed immunoblotting detection using anti-HAL, anti-VP1, and anti-PPDK. A vacuolar H+-pyrophosphatase (PPase) assay was carried out by measuring the AMDP-sensitive Pi release activity in the isolated fractions. Pi release was quantified using a malachite green assay (43 (link)). Ammonia release in acidocalcisome-enriched vesicles was performed in the presence/absence of l -histidine using an ammonia detection kit following the manufacturer’s instructions (Sigma-Aldrich).
Ammonia detection kit
Manufactured by Merck Group
Sourced in United States
The Ammonia detection kit is a laboratory equipment designed to quantify the concentration of ammonia in a sample. The kit provides a reliable and accurate method for the analysis of ammonia levels in various applications.
Automatically generated - may contain errors
2 protocols using ammonia detection kit
1
Isolation and Analysis of Acidocalcisomes
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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