How to prepare samples for peptide liquid chromatography?
Peptide liquid chromatography (Liquid Chromatography, abbreviated as LC) is a commonly used method for peptide separation and purification. The sample preparation method may vary depending on the specific experimental purpose and sample properties. Therefore, in actual practice, it is recommended to refer to relevant literature and experimental protocols to optimize the sample preparation method. The basic steps for preparing samples are as follows:
1. Sample Dissolution:
First, dissolve the peptide sample in a suitable solvent. Generally, water, acetonitrile, methanol, or a buffer containing a certain proportion of organic solvent can be used to dissolve peptides. The choice of solvent mainly depends on the properties of the peptide, such as its solubility and stability. It is important to ensure that the chosen solvent is compatible with the mobile phase of the liquid chromatography system.
2. Protein Treatment:
If there are impurity proteins in the sample, they can be removed by methods such as heating, acid treatment, or enzyme treatment. The treated sample needs to be adjusted to an appropriate pH value before proceeding to the next step.
3. Precipitate Removal:
To remove precipitates and large particle impurities from the sample, centrifugation can be performed. Typically, centrifugation at 10,000-15,000g for 10-15 minutes is sufficient. After centrifugation, transfer the supernatant to a new centrifuge tube.
4. Membrane Filtration:
To further remove fine impurities, microfiltration membranes (such as 0.22μm or 0.45μm) can be used to filter the sample. This helps to avoid clogging of the chromatography column and improves the effectiveness of chromatographic separation.
5. Concentration and Desalting:
If the sample contains excessive salts or solvents, methods such as centrifugal concentrators or gel filtration columns can be used for concentration and desalting. The treated sample can be diluted with the mobile phase.
6. Sample Volume Adjustment:
Adjust the processed sample to an appropriate volume and record the sample concentration. Then, dilute the sample according to the requirements of the liquid chromatography system.
7. Sample Storage:
Store the prepared sample under low temperature conditions (such as -20°C or -80°C) to minimize sample degradation.
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SDS-PAGE Protein Purity Analysis
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