Research Guide
How to Reconstitute Peptides: Complete Step-by-Step Guide
Peptide reconstitution is the process of dissolving lyophilized (freeze-dried) peptide powder back into a liquid solution for research use. Proper reconstitution technique is essential for maintaining peptide integrity, ensuring accurate concentrations, and achieving reproducible research results. This guide covers everything from workspace preparation to storage of reconstituted solutions.
What You Need
Before reconstituting any peptide, gather all required materials: the lyophilized peptide vial, bacteriostatic water (BAC water) as the reconstitution solvent, sterile syringes with appropriate needle gauges (typically 29-31G), alcohol swabs for sterilization, and sterile gloves. Work in a clean environment — ideally a laminar flow hood for research settings, or at minimum a sanitized, low-traffic workspace.
Step 1: Bring Materials to Room Temperature
Remove the lyophilized peptide vial and bacteriostatic water from cold storage. Allow both to equilibrate to room temperature for 15-20 minutes before proceeding. This step is critical — adding cold solvent to cold peptide powder can cause aggregation and incomplete dissolution. Opening cold vials also introduces moisture from condensation, which can degrade the remaining lyophilized powder.
Step 2: Sterilize Vial Tops
Clean the rubber stopper on both the peptide vial and the bacteriostatic water vial with a fresh alcohol swab. Allow the alcohol to air-dry completely before piercing the stopper. This prevents introduction of bacteria or contaminants into either solution.
Step 3: Withdraw Bacteriostatic Water
Using a sterile syringe, withdraw the desired volume of bacteriostatic water. Common reconstitution volumes are 1 mL or 2 mL, depending on the desired concentration. The volume you choose determines the concentration of your final solution — using less water creates a more concentrated solution, and vice versa. For example, adding 1 mL of BAC water to a 5 mg peptide vial creates a 5 mg/mL solution. Adding 2 mL creates a 2.5 mg/mL solution.
Step 4: Add Water to Peptide Vial (Slowly)
This is the most critical step. Insert the needle through the rubber stopper at an angle so the water runs down the inside wall of the vial. Do NOT inject water directly onto the lyophilized powder cake. Do NOT squirt or force the water in. Let it drip slowly along the glass wall. This gentle approach prevents agitation that could damage peptide bonds and cause foaming, aggregation, or denaturation. Never shake or vortex a peptide vial.
Step 5: Allow Dissolution
After adding the water, set the vial upright and allow the peptide to dissolve naturally. Most peptides dissolve within 5-10 minutes without any agitation. If needed, you can gently roll the vial between your palms — never shake it. The solution should become clear. If it remains cloudy or contains visible particles, this may indicate degradation or contamination.
Step 6: Storage of Reconstituted Peptide
Once reconstituted, store the peptide solution at 2-8°C (standard refrigerator) for short-term use (up to 3-4 weeks). For longer storage, aliquot the solution into smaller volumes and store at -20°C. Avoid repeated freeze-thaw cycles — each cycle can degrade the peptide by 5-10%. The bacteriostatic agent in BAC water (typically 0.9% benzyl alcohol) helps prevent microbial contamination but does not indefinitely preserve the peptide.
Need help with calculations?
Open Peptide CalculatorFrequently Asked Questions
What is the best solvent for reconstituting peptides?▼
Bacteriostatic water (BAC water) is the standard solvent for peptide reconstitution. It contains 0.9% benzyl alcohol as a preservative, which inhibits microbial growth and allows multiple withdrawals from the same vial. Sterile water can also be used but lacks the preservative, requiring single-use.
How much bacteriostatic water should I add?▼
The volume depends on your desired concentration. A common approach is 1 mL per 5 mg of peptide (creating a 5 mg/mL solution), but any volume from 0.5 mL to 3 mL can be used. Use a peptide calculator to determine the volume needed for your desired per-dose concentration.
Can I shake the vial to dissolve the peptide faster?▼
No. Never shake, vortex, or vigorously agitate a peptide vial. This can damage peptide bonds, cause aggregation, and create foam. Gentle rolling between palms is acceptable if needed. Most peptides dissolve within 5-10 minutes without any agitation.
How long does a reconstituted peptide last?▼
In bacteriostatic water, reconstituted peptides are generally stable for 3-4 weeks at 4°C and 3-4 months at -20°C. Stability varies by peptide — shorter peptides tend to be more stable. Avoid repeated freeze-thaw cycles.
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