Pepsin Solution Preparation
- Version: 2.0
- Author: Mingchuan Tang
- Last modified: 2025-09-09
Required Reagents
- Pepsin: Sigma P6887-250MG (250mg package) product link
- RNase free Water
- 12 M concentrated hydrochloric acid
- Activity: >3200 U/μL
- Optimal pH: 2–4
Preparation Steps
1. Prepare 1 M HCl
- Add 83 μL 12 M HCl to 917 μL RNase free Water (or 830 μL to 9.17 mL)
- Mix well
2. Prepare 0.01 M HCl
- Dilute 1 M HCl 100-fold: Add 10 μL 1 M HCl to 990 μL RNase free Water
- Mix well
3. Prepare 4 mg/mL pepsin storage solution (in 0.01 M HCl)
- Weigh pepsin powder and dissolve to 4 mg/mL with 0.01 M HCl
- Example: 4 mg pepsin + bring up to 1 mL with 0.01 M HCl
- Aliquot, store at 4°C for short term or at -20°C for long term
4. Working solution: 0.04% (w/v) pepsin (~1.4 U/μL) in 0.01 M HCl
- Dilute the 4 mg/mL storage solution 1:10 into 0.01 M HCl
- Example: 100 μL storage solution + 900 μL 0.01 M HCl → 1 mL at 0.04%
- Prepare fresh before use
5. Optional: 0.01% (w/v) pepsin (~0.3 U/μL) in 0.01 M HCl
- Further dilute 0.04% solution 1:4 with 0.01 M HCl
- Example: 250 μL of 0.04% + 750 μL 0.01 M HCl → 1 mL at 0.01%
Notes
- Target working solution: 0.04% (w/v) pepsin in 0.01 M HCl (10x from 4 mg/mL)
- Prepare working solutions fresh; avoid extended storage after dilution
- Use RNase free reagents for all operations
- Storage: pepsin powder at -20°C; 20 mg/mL storage solution aliquots at 4°C (short) or -20°C (long)
- Pepsin activity is optimal at pH 2–4; 0.01 M HCl keeps pH in the optimal range