What methods can be used to measure intracellular cysteine content?
To measure the intracellular content of cystine, the following methods can be used:
1. High-Performance Liquid Chromatography (HPLC)
- Sample Preparation: First, cystine needs to be extracted from the cells. This usually involves using freeze-thaw cycles and acidic or alkaline hydrolysis to break down the cells and release cystine.
- Derivatization: To enhance the detection sensitivity of cystine, it can be derivatized, for example, using o-phthaldialdehyde (OPA).
- Chromatographic Analysis: Using an HPLC system, separation is performed with an appropriate column and mobile phase, followed by detection with a UV or fluorescence detector.
2. Amino Acid Analyzer
- Sample Preparation: Similar to the HPLC method, cystine needs to be extracted from the cells.
- Electrophoretic Separation: Amino acid analyzers typically use ion-exchange chromatography for separation.
- Detection: Detection is performed through colorimetric or fluorescence reactions.
3. Mass Spectrometry (LC-MS/MS)
- Modern mass spectrometry techniques, especially liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used for the qualitative and quantitative analysis of cystine. This method is particularly useful for detecting intracellular metabolites, especially in metabolomics studies.
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Amino Acid Composition Analysis
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