How to Store Reconstituted Peptides?

Reconstitution is a common way of using lyophilized peptides. However, since water is introduced, the compound is now subject to degradation. But, with proper storage, the reconstituted peptides’ integrity can be extended.

This post will explain how this can be achieved.

Understanding Peptide Stability After Reconstitution

Peptides are composed of amino acids linked by peptide bonds. This property enables peptides to form structures that are chemically dynamic and environmentally responsive.

In their lyophilized (freeze-dried) state, peptides are relatively stable. This is because water, a key driver of degradation reactions, is absent. After reconstitution, however, molecular mobility increases. This could accelerate the degradation processes.

Below are some well-known degradation pathways that affect peptides in solution:

• Hydrolysis

Water has the ability to cleave peptide bonds. This may happen under extreme pH or elevated temperature conditions.

• Oxidation

Some amino acids are prone to oxidative modification. These are methionine, cysteine, and tryptophan.

• Deamidation

Some amino acids could undergo structural changes over time. Asparagine and glutamine residues are perfect examples.

• Aggregation

Peptides can self-associate or bind to certain surfaces. This property can alter effective concentration.

• Adsorption

Binding to glass or plastic surfaces may reduce measurable peptide levels

Choosing the Right Solvent Matters

Before we explain appropriate storage temperatures, it will be better to consider solvent choice first. This is because the solution used for reconstitution directly impacts peptide stability.

Common laboratory solvents are:

• Sterile water
• Buffered solutions (Such as phosphate-buffed saline or acetate buffers)
• Dilute acids (e.g., acetic acid)
• Organic solvents like DMSO (when solubility requires)

The pH of the solution is another critical variable. Remember that extremely acidic or alkaline conditions can speed up hydrolysis or deamidation. For some sequences, mildly acidic conditions may enhance stability. However, this will vary depending on the amino acid composition and intended research application. [2]

Another factor that affects stability is ionic strength. This explains why some peptides are more stable in low-salt environments while others require buffering for experimental stability.

As a general practice, we recommend:

• Using the minimum solvent volume necessary
• Ensuring solvent compatibility with downstream assays
• Maintaining a sterile technique when appropriate
• Avoiding unnecessarily transferring solutions between containers

After reconstitution, the countdown on solution stability begins. Thus, storage conditions must be optimized accordingly.

Optimal Temperature for Reconstituted Peptides

For some obvious reasons, temperature control is one of the most critical variables to preserve peptide integrity.

Short-Term Storage (2-8°C)

If you foresee a short-term use, refrigeration at 2-8°C is acceptable within controlled laboratory environments. This range is believed to slow down degradation reactions without resorting to freezing.

However, refrigeration is generally recommended for limited periods. It may be applicable for a few days or weeks. It will still depend on sequence and solvent composition.

If you plan to refrigerate peptides, they must be clearly labeled with:

• Reconstitution date
• Concentration
• Solvent used
• Storage temperature

NOTE: Even under refrigeration, peptides are susceptible to hydrolysis and oxidation over time.

Long-Term Storage (-20°C or -80°C)

If extended storage is needed, freezing is highly recommended:

• – 20°C is suitable for several standard research applications
• – 80°C can provide enhanced stability for highly sensitive or oxidation-prone sequences

Lower temperatures can decrease molecular motion. This significantly helps slow chemical reactions such as hydrolysis and deamidation. Some research workflows may require high reproducibility over extended timelines. In such a situation, ultra-low temperature storage is often preferred.

However, freezing introduces another important consideration: freeze-thaw cycling.

Avoiding Freeze–Thaw Damage Through Aliquoting

Repeated freeze-freeze thaw cycles can have a negative effect on peptide integrity.  This is because each cycle introduces the following:

• Thermal stress
• Potential condensation
• Increased risk of aggregation and precipitation

The most effective strategy to prevent these from happening is aliquoting.

Best Practices for Aliquoting

1. Divide the reconstituted peptide into single-use or limited-use volumes. Do this step immediately after preparation.
2. Use low-binding microcentrifuge tubes to reduce surface adsorption.
3. Minimize headspace within tubes to limit oxygen exposure.
4. Label each aliquot clearly with relevant information.

Aliquoting ensures that only the amount needed for an experiment is thawed. By doing so, this practice preserves the remainder in stable frozen conditions.

Protecting Peptides from Environmental Stressors

Here, temperature is not the only concern. Reconstituted peptides also need protection from other environmental stressors.

Light Exposure

Certain amino acids display sensitivity to ultraviolet and visible light. These could include tryptophan and tyrosine. Prolonged light exposure may introduce structural changes or oxidation.

To mitigate this:

• Storage solutions in amber or opaque containers when appropriate.
• Minimize time under laboratory lighting during handling.
• Avoid placing samples in direct light sources.

Oxidation

Oxygen exposure is another identified environmental stressor to look out for. Indeed, not all peptides require special atmospheric control. However, highly oxidation-prone sequences may benefit from added precautions.

Some of the best practices are:

• Keeping containers tightly sealed.
• Minimizing repeated opening of tubes.
• Using inert gas overlays (e.g., nitrogen) in specialized research settings as warranted

Surface Adsorption

Peptides can adsorb to glass or other plastic surfaces. Such a situation effectively reduces usable concentration.

To reduce adsorption:

• Use low-retention labware.
• Avoid unnecessary transfers between containers.
• Prepare solutions at appropriate concentrations to minimize surface-area effects.

Monitoring Stability and Detecting Degradation

Even with careful storage, it is still highly advisable to observe periodic observation. Signs that degradation may be occurring include:

• Cloudiness or visible precipitation
• Unexpected color changes
• Inconsistent assay performance
• Reduced signal intensity in analytical measurements

If there is a need for confirmation, laboratories could resort to various analytical techniques:

• High-performance liquid chromatography (HPLC)
• Mass spectrometry
• UV absorbance profiling
• Gel electrophoresis (where appropriate)

Routine monitoring is necessary to support data reliability. It even ensures that experimental outcomes are influenced by intended variables.

Supporting Reliable Research Through Proper Storage

Reconstituting a peptide is not the last step in peptide preparation. Rather, it marks the beginning of a new stability phase. Once in a solution, peptides will require deliberate environmental control. The goal is to maintain its structural integrity.

This post has shown you the various ways researchers can significantly extend peptide usability. Plus, each step safeguards experimental accuracy.

Proper storage is not just about preservation. It is also about protecting the integrity of the research itself.

References:

1. Forbes, J., & Krishnamurthy, K. (2023, August 28). Biochemistry, Peptide. PubMed; StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK562260/
2. Adav, S. S. (2025). Advances in the Study of Protein Deamidation: Unveiling Its Influence on Aging, Disease Progression, Forensics and Therapeutic Efficacy. Proteomes, 13(2), 24–24. https://doi.org/10.3390/proteomes13020024
3. Zapadka, K. L., Becher, F. J., Gomes dos Santos, A. L., & Jackson, S. E. (2017). Factors affecting the physical stability (aggregation) of peptide therapeutics. Interface Focus, 7(6), 20170030. https://doi.org/10.1098/rsfs.2017.0030