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How do I quantify the final peptide concentrations?
Posted September 30, 2024
Affinity ChromatographyCalculating ConcentrationChromatographic techniquesMeasure absorbanceProtein Assays & AnalysisProtein PurificationProtein concentrationmolecular weightspectrophotometer
Answer
- Purify the Peptide:Purify the crude peptide using reverse-phase High-Performance Liquid Chromatography (HPLC) to isolate the target peptide sequence.
- Lyophilize the Peptide:After purification, lyophilize the peptide into a powder. This powder may contain non-peptide substances like water, solvents, salts, and counter ions.
- Prepare the Spectrophotometer: Use a suitable blank to calibrate the spectrophotometer. This involves setting the instrument to zero absorbance using a blank solution that matches the sample buffer.
- Measure Absorbance:
- Wavelengths: Measure the absorbance of the peptide solution at two wavelengths: 215 nm and 225 nm.
- Absorbance Check:Ensure that the absorbance at 215 nm is less than 0.5. If it is higher, dilute the sample with a buffer and measure again.
- Calculate concentration: Determine mg/mL: Use the formula: mg/mL = (A215 − A225) × df × 0.144 mg/mL = (A215 − A225) × df × 0.144
- Convert to Molar Concentration:
- Find Molecular Weight:Divide the mg/mL value by the molecular weight (MW) of the peptide to get the molar concentration in moles per liter (M).
- Convert to µM:Multiply the result by 1,000,000 to convert to micromolar (µM) concentration.