Storage and Reconstitution: A Bench Protocol

Scope, and the six hazards this protocol controls

The protocol covers lyophilised research peptides in sealed vials and the solutions made from them for laboratory work. It gives no instructions for administration to people or animals.

No calendar date decides when a peptide is spent. It is worn down by a handful of chemical and physical processes, and each step below is there to hold one of them back.

Temperature table

The table gives no day counts: how long a peptide lasts is set by the solvent, the pH, its salt form and the sequence itself, so stability figures measured for that peptide beat any rule of thumb.

Part A: from parcel to freezer

Dry peptide in a sealed vial degrades slowly, which is why a normal courier journey at ambient temperature does it no harm. King Peptides, the shop that the order links on this site lead to, dispatches from the Netherlands in a tracked parcel: usually 1–2 business days within the Netherlands and 3–5 business days to Belgium and Luxembourg, with no customs clearance inside the EU. Delivery and paperwork are treated in the Benelux buying guide.

Heat that lasts is the real concern. A parcel that sits for days in a hot delivery van, or in a sun-baked letterbox in midsummer, has had a harder journey than one that waited overnight at a depot. Peptide solutions tolerate warm transit far worse, which is one reason research peptides are sent dry.

1. Unpack on the day of delivery. A desk is not a storage place.
2. Inspect the contents through the glass. Expect a dry cake or a loose, free-moving powder. Material that has shrunk, turned sticky or gone glassy is a warning sign.
3. Check the lot number on every vial against its certificate of analysis. Checking a Certificate of Analysis, Line by Line explains what else that document should show.
4. Mark every vial with three things: what it is, its lot number and the date it came in.
5. Put the vials in a lidded box with a sachet of desiccant, keep the box dark and freeze it at −20 °C.

Part B: keeping and opening the dry powder

No form of a peptide is more stable than the freeze-dried one. Hold it at −20 °C in a closed vial, away from light and humidity. An archive intended for the long term does better still at −80 °C. Frost-free freezers are a poor choice for long storage, because their automatic defrost cycle lets the temperature rise for a short time at regular intervals. A manual-defrost unit stays steadier.

Opening is the risky moment. Glass that comes out at −20 °C is far colder than room air, so water condenses on the outside at once, and on the inside the second the stopper is off.

1. Remove the vial, still closed, and leave it until it is at room temperature. A desiccator is the ideal place for this.
2. Open it, do the work without delay and reseal.
3. Return the vial to its box and the box to the freezer.
4. If many sessions are planned with one lot, split it once, at the first opening, into single-use portions: pre-weighed powder, or a solution frozen in aliquots.

Part C: solvent choice and reconstitution

Pick the solvent for the peptide and for the purpose of the solution.

• Bacteriostatic water. Sterile water with 0.9% benzyl alcohol added as a preservative. Because the alcohol keeps bacteria in check, the same vial can be entered many times over a longer span.
• Sterile water, no preservative. For a solution that is used immediately or goes straight into frozen aliquots, and for experiments that benzyl alcohol might disturb, some cell-based assays for example.
• Something stronger. A very hydrophobic or heavily charged sequence may dissolve only in dilute acid, in a buffer or with a little organic solvent. Consult the solubility notes from the supplier first.

1. Let the closed vial and the solvent warm to room temperature.
2. Clean the work surface and lay out sterile equipment. The stopper gets a wipe with 70% alcohol before it comes off or anything goes through it.
3. Measure the volume worked out in Part D with a calibrated pipette, never by eye: the relative error grows as the volume shrinks.
4. Let the solvent run slowly down the glass wall. It should not land on the cake directly.
5. Dissolve by gentle swirling, or by rolling the vial between the fingers. Shaking is ruled out: it drives air into the liquid, and peptides aggregate where air meets water.
6. Inspect against the light. A good solution is clear. Haze or particles mean that the peptide has not dissolved completely or has aggregated. Discard a solution that goes cloudy or changes colour later on.
7. Write on the vial: peptide, concentration, solvent, date, lot number.

Part D: concentration arithmetic

Stock concentration equals the mass of peptide divided by the volume of solvent. Put the sums on paper each time, because losing a factor of ten is the classic error.

To take the bottom row further: 100 µl of that stock carries 500 µg of peptide. Mixing 100 µl of it with 900 µl of buffer is a tenfold dilution and gives a working solution of 0.5 mg/ml.

Assays are normally planned in molar units. Molarity is the mass concentration divided by the molecular weight, so the heavier the molecule, the fewer moles the same mass contains. A 5 mg/ml stock of BPC-157 (1419.5 Da) is about 3.5 mM. The same mass concentration of MOTS-c (2174.6 Da) is about 2.3 mM.

One correction remains. Find out what the 10 mg on a label refers to. When it is powder weight, multiply by the net peptide content first, a figure the certificate guide explains. With a content of 80%, 8 mg of peptide are present, and the bottom-row stock comes out at 4 mg/ml instead of 5.

Part E: holding a solution

In water a peptide loses most of the protection it had as a powder: hydrolysis and deamidation have a medium, oxygen is dissolved throughout, and bacteria can multiply. While a solution is in use, keep it at 2–8 °C, out of the light, and finish it soon. How soon varies with the sequence, the solvent, the pH and the number of times the vial is entered, so this protocol names no number of days.

1. Decide at reconstitution whether any of the stock must outlast short-term use. If so, portion it into single-use aliquots straight away.
2. Freeze the aliquots at −20 °C or −80 °C.
3. Allow one thaw per aliquot and throw away what is left over. A tube that goes in and out of the freezer loses peptide quietly.
4. Put dilute solutions in low-binding tubes. At low concentration a measurable fraction of the peptide adsorbs to ordinary plastic and to glass.
5. Stay below about pH 8. Alkaline conditions speed up deamidation and the oxidation of cysteine.
6. A preservative-free solution that is to be kept at all must be sterile-filtered or made up aseptically.

Sequences that need extra care

Oxidation problems trace back to three residues. Methionine gains an oxygen atom and becomes the sulfoxide, which is 16 Da heavier and may differ in activity. Cysteine pairs up into disulfide bonds, within one chain or across two. Tryptophan is oxidised by air, and light hastens it.

For these entries, tighten the general rules. Leave as little headspace as possible above powder and solution, and store vials in darkness. Make up solvents fresh or degas them, and think about blanketing stored aliquots with nitrogen or argon. DMSO (dimethyl sulfoxide) is unsuitable for sequences containing methionine or cysteine, since the solvent itself is able to oxidise them.

GHK-Cu, a copper complex that owes its blue colour to the bound metal, has rules of its own. Darkness is mandatory for it, dry or dissolved. EDTA and other chelating agents must stay out of its buffers, because they compete with the peptide for the copper and can pull the metal away. If a GHK-Cu solution changes colour, assume that the complex has changed too.

Quick answers

Is a laboratory fridge cold enough for unopened vials? For a brief stay, yes. At 2–8 °C the powder is protected, but much less than in a freezer, so anything longer belongs at −20 °C.

The parcel travelled unrefrigerated for several days. Is the material still usable? Most likely, as long as the powder looks dry and intact and the trip was a matter of days, not weeks. Freeze the vials as soon as they arrive.

The solution stays cloudy after swirling. What now? Either some peptide is still undissolved or aggregation has begun. Gentle swirling at room temperature can help. If the haze persists, a different solvent is probably needed; see the solubility notes from the supplier.

How long does a reconstituted peptide keep in the fridge? No single figure fits every peptide; sequence, solvent, pH and handling all play a part. Plan for a short period at 2–8 °C and freeze single-use aliquots for anything beyond that.