Dimitar Marinov, Ph.D.
Dimitar Marinov, Ph.D.Looking for a detailed guide on how to reconstitute peptides for research?
Then look no further, as this guide will provide detailed information on how to mix and store peptides for research experiments.
Many peptides are available as reference materials in the form of lyophilized powders. Thus, researchers who plan to incorporate peptides into their work must know how to properly reconstitute them into liquids.
For researchers, this comprehensive review will provide essential information on what solvents to use, how to calculate the appropriate quantities for reconstitution, and a step-by-step guide on mixing peptides in research settings.
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What are Peptides?
At the most basic level…
Peptides are molecular structures composed of amino acids — the same building blocks that make proteins. Peptides and proteins are formed when amino acids link together through peptide bonds, creating an amino acid chain sequence.
But the main difference between the two is the length and complexity of their chains. Peptides are shorter, usually made of 2-50 amino acids, and the majority have a linear structure, although there are likewise cases of non-linear or cyclic peptides [1].
In comparison, proteins are typically composed of one or more polypeptide chains that can consist of hundreds or thousands of amino acids and form a three-dimensional structure arising from interactions between different regions of the polypeptide chain [2].
Despite their less complicated structure, peptides play crucial roles in the body's biological processes.
They can act as signaling molecules, transmitting messages between cells and coordinating various physiological functions. They can influence hormone production, immune responses, and cell growth and repair [3].
Due to their diverse functions, peptides have gained attention in medicine and research. They are used to develop new drugs and therapies, as they can target specific receptors and modulate biological pathways [4].
What Does It Mean to Reconstitute Peptides?
Simple…
Reconstituting peptides refers to the process of dissolving or rehydrating lyophilized (freeze-dried) peptides to prepare them for use in research.
This step is required because research peptides cannot exert their biological activity in a solid state and must be reconstituted back into a liquid state.
Most peptides are also in a liquid state when they are initially synthesized and manufactured. However, liquid formulations tend to be unstable due to their physical and chemical degradation susceptibility.
This is why manufacturers turn them into a solid state via various methods, such as freeze-drying. More specifically, freeze-drying is also known as lyophilization and turns the peptide into a dry, powder form to enhance its shelf life [5].
A lyophilized peptide powder is more stable and can be stored for longer periods without degradation. Lyophilization helps preserve a peptide’s integrity and activity, making it easier to handle, store, and transport [6].
Thus, when ordering peptides online, researchers will receive products in a dry, powdered form, which guarantees that they are shelf-stable and still viable for experiments.
These “raw” peptides must be reconstituted back into a liquid form using an appropriate solvent to exert their biological activity in experimental settings.
The most common solvents for reconstitution include bacteriostatic water, sterile water, and organic solvents like dimethyl sulfoxide (DMSO) [7].
The most appropriate solvent will depend on several factors, such as the type of peptide, the research purpose, and desired shelf-life.
For example, most peptides dissolve well in bacteriostatic or sterile water. However, poorly soluble polar and non-polar molecules will require organic solvents such as DMSO [8].
It is important to note that reconstituted peptides should be handled with care to maintain their stability. Factors such as extreme temperature, pH, and exposure to light should be avoided to prevent denaturation and loss of biological activity [9, 10].
Further, following established protocols and guidelines specific to the peptide being used is crucial to ensure accurate and reproducible results in research.
Keep reading to find a detailed step-by-step guide on how to reconstitute peptides for research purposes.
How to Reconstitute Peptides
Before we go over the reconstitution process step-by-step, researchers must ensure they have all the supplies needed for the process.
To reconstitute research peptides correctly, researchers will need the following materials:
- Vial of lyophilized peptide powder (“raw” peptide)
- Vial of a sterile solvent, such as bacteriostatic water
- Alcohol prep pads
- A sterile syringe of at least 3cc
- Disposable sharps container
How to Mix Peptides | A Step-by-Step Guide
Once researchers have all essential materials, follow this step-by-step guide on how to reconstitute research peptides:
- Before starting, allow the peptide and bacteriostatic water vials to reach room temperature for 30 minutes, away from direct light or heat sources.
- Start by disinfecting the stoppers of both vials using alcohol prep pads.
- Assemble the large sterile needle and syringe and draw in about 1mL of air. Then insert the needle into the vial with the sterile solvent.
- Inject the air inside the vial to prevent negative pressure and immediately withdraw the correct amount of sterile solvent needed for reconstitution (usually 1mL).
- Pull the needle out of the solvent and insert it into the vial with lyophilized peptide.
Then drip or slowly inject the solvent from the syringe while aiming the needle tip at the vial wall. To prevent foaming, do not aim directly toward the powder or spray the solvent. - Once researchers have injected the correct amount of solvent, they can dispose of the needle and syringe in a sharps container.
- Let the peptide dissolve naturally within the solvent. In addition, researchers can use sonication if available or very gently roll/swirl the vial. Avoid forceful tapping or shaking, as this can damage the peptide structure. Also, avoid tapping the syringe before injection.
- After the peptide has dissolved, check the clarity of the liquid and look for any particles. If the solution is cloudy or if there are any particles, discard it.
Once the peptide is reconstituted, refer to the specific product label for accurate dosage and storage instructions, if any.
Peptide Reconstitution Calculator and Chart
When reconstituting peptides for research, scientists must start by getting acquainted with the quantity of the lyophilized peptide contained in the vial. This is a fixed amount measured in milligrams (mg) or micrograms (mcg), as stated on the label.
The amount of solvent used will determine the volume of the reconstituted peptide to be withdrawn for a desired dose.
For instance, if a researcher adds 2mL of a solvent to a peptide, they will need to inject twice the volume compared to another researcher who wants to administer the same dose but adds only 1mL.
Some researchers may find it difficult to calculate the appropriate amount of peptide to administer because, unlike reconstitution, the administration process typically requires smaller insulin syringes for subcutaneous administration.
Insulin syringes come in various sizes, typically ranging from 0.3ml to 2.0ml. However, the tick marks on insulin syringes represent numbers of units (insulin units) rather than milliliters. They are usually labeled as either U-40 or U-100, indicating the type of insulin they are designed for.
To avoid confusion, the expert team at Peptides.org has created a comprehensive calculator that will help researchers easily determine the number of units to administer per dose based on the amount of the peptide and the quantity of solvent used for reconstitution.
More info here…
Peptides Dosage Calculator
With that calculator, all researchers have to do is follow these 5 simple steps:
- Select the syringe size available and plan to use for administering the peptide, such as 0.3mL, 0.5mL, or 1mL.
- Double-check the peptide's label to find out its specific amount. Then, input the mass of the peptide powder as indicated on the vial, usually in milligrams (mg).
- Input the amount of solvent injected into the vial to reconstitute the peptide, typically in milliliters (ml).
- Last, select the peptide dose that researchers wish to administer with each injection. Keep in mind t0 enter the amount in micrograms (mcg). If researchers plan on administering milligrams of the peptide per injection, then they must convert them into mcg by multiplying the mg x 1000.
- At the bottom, the calculator will automatically show how many tick marks must be withdrawn from the reconstituted peptide to deliver the desired dose via an insulin syringe.
By following these steps and using the reconstitution calculator, researchers can ensure proper dosage and reconstitution of peptides for success in experimentation.
Bacteriostatic Water vs. Sterile Water
Two of the most commonly used solvents for dissolving research peptides are bacteriostatic water and sterile water.
In general, bacteriostatic water is the preferred option as it contains 0.9% benzyl alcohol, which prolongs the peptide’s shelf-life [11].
The benzyl alcohol has no toxicity at these concentrations, while it can effectively suppress the growth of microorganisms. Due to the presence of the preservative, the pH of bacteriostatic water is typically around 5.7 (4.5 to 7.0), which also helps improve the stability of most peptides [12, 13].
Bacteriostatic water is typically supplied in plastic vials made of specially formulated polyolefin, which has confirmed safety in animal tests. The multiple-dose vials allow repeated withdrawals of bacteriostatic water for the reconstitution of peptides [14, 15].
Unopened and unused bacteriostatic water has a shelf life of over two years when stored properly. After breaching the safety seal and opening the vial, bacteriostatic water has a shelf-life of up to four weeks if refrigerated properly at 36 to 46 degrees F (2 to 8 degrees C).
Similarly, peptides reconstituted with bacteriostatic water have a four-week shelf life when refrigerated at these temperatures.
A downside of bacteriostatic water is that some test subjects may be allergic to benzyl alcohol [16]. In such cases, subjects should receive only peptides reconstituted with sterile water.
In comparison, sterile water does not suppress microbial growth, and its shelf-life is only 24 hours after opening. Any peptide reconstituted with sterile water also becomes unsuitable for use after 24 hours, even if refrigerated.
However, sterile water is the only viable option for test subjects with hypersensitivity to benzyl alcohol.
How to Store Reconstituted Peptides
Research peptides that are reconstituted with bacteriostatic water must be refrigerated at 36 to 46 degrees F (2 to 8 degrees C) and under these conditions remain viable for up to four weeks.
Avoid freezing reconstituted peptides as this can impact the compound’s integrity and functionality. This is because ice crystal formation can disrupt the peptide structure, potentially leading to loss of biological activity or altered properties.
Additionally, freeze-thaw cycles can exacerbate the damage caused by freezing and lead to aggregation or precipitation.
In cases where freezing is absolutely necessary, the United Kingdom National Institute for Biological Standards and Control (NIBSC) recommends that researchers freeze and store aliquots of the peptide at -4°F (-20°C) or lower temperatures [17].
To minimize potential damage, it may be beneficial to use slow freezing methods. Consequently, fast thawing may help reduce the number of ice crystal formations, although the optimal strategy may vary from one compound to another [18].
It is likewise important to avoid multiple freeze-thaw cycles as this leads to cumulative damage. The better alternative would be freezing the reconstituted research peptides (any amount that will not be used within four weeks) as several aliquots in empty sterile vials and then thawing them one by one.
Proper storage conditions, such as using cryoprotectants or stabilizing agents, can further help mitigate damage during freezing and subsequent storage [19].
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How to Store “Raw” Peptides
“Raw” peptides are typically shipped in the form of dry lyophilized powders that are sealed within sterile vials, usually under an atmosphere of dry inert gas.
This significantly extends the shelf life of lyophilized peptides to several years when stored properly in a dark, cool, and dry place.
The shelf-life can be further extended by storing the peptides at low temperatures, especially under -4°F (-20°C).
Freezing them is generally safe as they do not contain any water molecules, and there is no risk of ice crystal formation.
Before reconstitution, “raw” peptides should be left at room temperature. Neither lyophilized nor reconstituted peptides should be exposed to heat or direct sunlight.
FAQ | How to Reconstitute Peptides
The field of peptide research includes a wide range of studies and directives related to peptide reconstitution.
However, there tends to be a concentration of inquiries in a select few areas of interest. Here is a compilation of the most frequently asked questions to cater to these specific interests:
How long do peptides last once reconstituted?
The shelf-life of reconstituted peptides depends on the solvent. Peptides reconstituted with bacteriostatic water last for up to four weeks when refrigerated at 36 to 46 degrees F (2 to 8 degrees C).
How much bacteriostatic water should be mixed with peptides?
Peptides for research are usually reconstituted with 1mL of bacteriostatic water per vial. Larger doses can also be used, although this increases the respective volume needed to deliver the same peptide dose per injection. Higher injection volume may lead to increased discomfort, especially for subcutaneous injections.
Can researchers mix peptides in the same syringe?
Not all peptides can be mixed in the same syringe, as some peptides may undergo chemical reactions with one another. Therefore, make sure to mix only peptides that are already available for purchase as peptide blends from reputable vendors.
How do researchers store sterile water for injections?
Sterile water can be stored for prolonged periods of time before opening. Once opened, there is a high risk for bacterial contamination and it expires within 24 hours. Peptides reconstituted with sterile water also expire within 24 hours, even if refrigerated.
Is distilled water the same as sterile water?
No, distilled water is sterile water that is also devoid of any minerals or electrolytes and is hypotonic for the human body. Regardless, injecting small amounts of distilled, sterile or bacteriostatic water cannot disturb the electrolyte balance and homeostasis in adults.
How to reconstitute BPC-157?
BPC-157 is available as reference material from reputable retailers such as Limitless Life in vials of 5mg.
Human studies with the peptide are lacking, but animal research suggests that it can be injected in doses of up to 10mcg/kg in rats, which extrapolates to about 1.61mcg/kg in humans, using the practice guide for dose conversion by Nair et al. (2016) [20, 21].
Therefore, researchers may consider adding 1mL, 2ml, or 3ml of bacteriostatic water to a 5mg BPC-157 vial, which would mean that they will have to withdraw 4U, 8U, or 12U respectively, via U-100 insulin syringes for an estimated dose of 200mcg BPC-157.
How to reconstitute TB-500?
TB-500 is another peptide that can be purchased as reference material from reputable online vendors. It typically comes in 5mg and 10mg vials.
Unfortunately, the available human studies have used TB-500 in the form of eye drops or pre-treated transplants, and therefore, there is no safe or recommended dosage [22, 23].
Gray literature sources report doses in the range of 5-10mg per week. If researchers consider using 1mL of bacteriostatic water for reconstituting a 10mg TB-500 vial, then they will have to withdraw 20U via an U-100 insulin syringe for an estimated dose of 2mg.
How to reconstitute tirzepatide?
Tirzepatide is a novel dual-incretin mimetic that is approved for therapy in patients with type 2 diabetes and shows potent weight loss effects, according to research [24].
In addition, tirzepatide is available as reference material for research purposes, and qualified researchers can purchase it from trusted sources. For example, Limitless Life offers 5mg vials for reconstitution.
When using the available research as a reference, the recommended starting dose of tirzepatide is 2.5mg/weekly, and it can be increased every four weeks up to a maximum of 15mg/weekly [24].
Researchers who have used 1mL of bacteriostatic water for reconstituting the 5mg vial will have to withdraw 50U via an U-100 insulin syringe to initiate their experiments with 2.5mg/weekly.
How to reconstitute semaglutide?
Semaglutide is an incretin mimetic approved for numerous indications such as weight loss in overweight and obesity, glycemic control in type 2 diabetes, and more. It is also the only incretin mimetic approved in both oral and injectable formulations [25, 26].
Semaglutide is also available as a reference material for researchers who plan on conducting experiments with this research peptide.
Using the available research as a reference, the recommended starting dose of semaglutide is 0.25mg/weekly, and it should be increased every four weeks up to a maximum of 2.4mg/weekly [25, 26].
Researchers who have used 1mL of bacteriostatic water for reconstituting the 3mg vial will have to withdraw 8.3U via an U-100 insulin syringe to initiate their experiments with 0.25mg/weekly.
Peptide Reconstitution | Verdict
In conclusion, research peptides hold significant promise in advancing pharmaceutical and medical fields, attracting the attention of prominent researchers worldwide.
Thus, handling and reconstituting these reference materials correctly is crucial to ensure successful experimentation.
The guide above offered comprehensive and evidence-based instructions on peptide reconstitution, suitable solvents, and proper storage methods.
When obtained from a trusted vendor, research peptides can be safely handled and analyzed by qualified laboratory professionals.
David Warmflash, M.D.