Storing and Reconstituting Research Peptides

What degrades a peptide

Peptides do not simply expire on a date. They degrade through specific chemical and physical processes, and nearly every storage rule exists to slow one of them down. Knowing which process a rule targets makes it easier to judge the situations the rules do not cover.

• Moisture. Water allows hydrolysis of peptide bonds and deamidation of asparagine and glutamine. Lyophilised powder is hygroscopic and draws moisture from the air whenever a vial stands open.
• Heat. Every degradation reaction runs faster at higher temperatures. A few hours of warmth matter little; weeks of it matter a great deal.
• Light. Ultraviolet and strong visible light drive photo-oxidation, particularly of tryptophan, and can affect metal complexes such as GHK-Cu.
• Oxygen. Air turns methionine into methionine sulfoxide, joins free cysteines into disulfides and damages tryptophan.
• Freeze–thaw cycles. As a solution freezes, peptide and salts concentrate in the shrinking liquid phase, which can drive aggregation and chemical change. Each cycle adds to the damage.
• Microbes. A solution without a preservative can support bacterial growth once its vial has been opened.

Storing lyophilised powder

Freeze-dried powder is the most stable form a peptide can take. The standard condition is −20 °C, in the dark and dry, in a closed vial. For archiving material over long periods, −80 °C slows degradation further. Freezers with an automatic defrost cycle are less suitable for long storage, because they warm up briefly at regular intervals; a manual-defrost freezer holds a steadier temperature.

Most of the damage done to stored powder happens at the moment of opening. A vial taken straight from the freezer is colder than the room, and moisture condenses on it and inside it as soon as the stopper comes off. Let the closed vial reach room temperature first, ideally in a desiccator, then work quickly, close it and return it to the freezer. Where material will be used over many sessions, preparing single-use portions early, as weighed powder or as frozen aliquots of a solution, avoids exposing the whole lot again and again.

Keep vials in a closed box with a desiccant sachet, out of the light, and label each one with the peptide, the lot number and the date it arrived. A dark box costs nothing and suits every peptide, but it matters most for tryptophan-containing peptides and for copper complexes.

What happens in transit

Lyophilised peptides generally tolerate a normal courier journey at ambient temperature. The reactions described above are slow in a dry powder, and a few days at room temperature in a sealed vial is not what shortens its life. The risk is prolonged heat: a parcel left for days in a hot van, or in a sunlit letterbox in midsummer, is a different matter from one that spends a night in a sorting centre.

Two practical points follow. Move vials into the freezer on the day they arrive rather than leaving them on a desk, and look at the powder before storing it: it should be a dry cake or loose powder, not a shrunken, sticky or glassy residue. Solutions, including premixed products, are far less tolerant of warm transit than powder is, which is one reason research peptides are shipped dry. Delivery times across Europe are covered in our guide to buying research peptides in Europe.

Storage at a glance

The table summarises conditions by form. The durations are deliberately qualitative: stability depends on the sequence, the salt form, the solvent and the pH, and measured stability data for a specific peptide always outrank a general rule.

Reconstitution in the laboratory

For most experiments the powder has to be dissolved first, and the solvent depends on the peptide and on how the solution will be used. Bacteriostatic water is sterile water containing 0.9% benzyl alcohol, a preservative that suppresses bacterial growth, so a vial can be sampled repeatedly over a longer period. King Peptides lists Bacteriostatic Water 10 ml in its catalogue alongside the peptides. Sterile water without preservative suits solutions that will be used at once or frozen in aliquots, and experiments in which benzyl alcohol could interfere, such as some cell-based assays. Very hydrophobic or strongly charged peptides may need a dilute acid, a buffer or a small amount of organic solvent; the solubility notes from the supplier are the place to start.

1. Let the closed vial and the solvent reach room temperature.
2. Work in a clean area with sterile equipment, and disinfect the stopper with 70% alcohol before opening or piercing it.
3. Add the measured volume of solvent slowly, down the inside wall of the vial rather than straight onto the powder.
4. Swirl or roll the vial gently until the powder has dissolved. Do not shake it: shaking whips air into the liquid and can make peptides aggregate at the air–water interface.
5. Check that the solution is clear. Cloudiness or visible particles point to incomplete dissolution or to aggregation.
6. Label the vial with the peptide, the concentration, the solvent, the date and the lot number.

A worked concentration example

The arithmetic is simple, but a factor of ten is easy to lose, so it pays to write it out every time. Suppose a vial contains 10 mg of peptide and 2 ml of solvent is added. The stock concentration is 10 mg ÷ 2 ml = 5 mg/ml, which is the same as 5 µg/µl. A 100 µl portion of that stock holds 500 µg. To prepare a working solution at 0.5 mg/ml, dilute the stock tenfold: 100 µl of stock plus 900 µl of buffer.

For assay work, molar concentration is usually more useful than mass per volume. Divide the mass concentration by the molecular weight. For BPC-157 at 1419.5 Da, 5 mg/ml is 5 g/l ÷ 1419.5 g/mol, or about 3.5 mmol/l (3.5 mM). The same 5 mg/ml of MOTS-c, at 2174.6 Da, comes to about 2.3 mM, because each molecule is heavier.

Two corrections keep the numbers honest. If the 10 mg on the label is the weight of the powder rather than of the peptide, net peptide content has to be taken into account: at 80%, the vial holds 8 mg of peptide and the stock is 4 mg/ml, not 5. Our guide to reading a certificate of analysis explains where that figure comes from. And measure the solvent with a calibrated pipette rather than estimating it, because the smaller the volume, the larger the relative error of a guess.

Storing peptide solutions

Once dissolved, a peptide is far less stable than it was as a powder. Water gives hydrolysis and deamidation a medium to work in, dissolved oxygen reaches every molecule, and microbes can grow. A solution in active use belongs at 2–8 °C, protected from light, and should be used up within a short period. How short depends on the peptide, the solvent, the pH and how often the vial is opened. There is no universal figure, and measured stability data outrank any rule of thumb.

For longer storage, divide the stock into single-use aliquots, freeze them at −20 °C or −80 °C, and thaw each one only once. Repeatedly freezing and thawing the same tube is an easy way to lose a peptide without noticing. Three further habits help.

• Low-binding tubes for dilute solutions. At low concentrations a noticeable share of the peptide can stick to plastic and glass surfaces.
• No alkaline conditions. A pH above about 8 speeds up deamidation and the oxidation of cysteine.
• Sterility for anything kept. Solutions made without a preservative should be sterile-filtered or prepared aseptically if they are to be stored at all.

Oxidation-prone and light-sensitive peptides

Three amino acids cause most oxidation problems. Methionine is converted to methionine sulfoxide, adding 16 Da, a change that can alter activity. Cysteine forms disulfide bonds, within a molecule or between two of them. Tryptophan oxidises in air and faster in light. On this site, MOTS-c contains two methionines and a tryptophan, Semax begins with a methionine, and GHRP-2, GHRP-6, hexarelin, melanotan II and PT-141 all carry tryptophan. Cysteine matters most for IGF-1 LR3, whose fold depends on three disulfide bonds. CJC-1295 shows the design side of the problem: it carries leucine at position 27, where natural GHRH has an oxidation-prone methionine.

For these peptides the general rules apply with extra care. Keep the air space above the powder or solution small, keep the vial dark, use freshly prepared or degassed solvents, and consider overlaying stored aliquots with nitrogen or argon. Avoid dimethyl sulfoxide (DMSO) as a solvent for peptides containing methionine or cysteine, because it can oxidise them.

Copper complexes need a note of their own. GHK-Cu owes its blue colour to the bound copper, and it should be kept strictly in the dark, as powder and in solution. Chelating agents such as EDTA compete for that copper and can strip it from the peptide, so they do not belong in its buffers. A change of colour in a GHK-Cu solution suggests the complex itself has changed.

Frequently asked questions

Can lyophilised peptides be kept in an ordinary fridge? For short spells, yes: 2–8 °C slows degradation, though far less than a freezer does. For anything beyond that, −20 °C is the standard condition.

Bacteriostatic water or sterile water? Bacteriostatic water, with its 0.9% benzyl alcohol, suits a solution that will be sampled repeatedly. Sterile water suits solutions used at once or frozen in aliquots, and assays in which benzyl alcohol could interfere.

My vial arrived at room temperature. Is the peptide damaged? Unlikely, if the powder is dry and intact and the journey took days rather than weeks. Lyophilised peptides generally tolerate a normal courier journey; move the vial to the freezer on arrival.

Why is my solution cloudy? The peptide has either not fully dissolved or has begun to aggregate. Gentle swirling at room temperature sometimes helps; if it does not, the peptide may need a different solvent, and the solubility notes from the supplier are the next place to look.