Q: What may happen during the peptide storage?

Some typical degradation reactions or racemization may include the oxidation of Met, Trp, Tyr, or Cys. The deamidation of Asn, Gln, and the C-terminal amide may happen. The aspartimide may form. There might be the cleavage of Asn-Pro. The N-terminal Gln may form pyroglutamine. The dimerization of Trp and Tyr may form.

Q: Why Reconstitute Peptides?

Reconstitution is the process of adding a solvent, usually bacteriostatic water, to the lyophilized peptide powder to prepare it for use. This process is essential because it allows for accurate dosing and helps preserve the peptide's stability

Q: What are Peptides?

Peptides are short chains of amino acids, the building blocks of proteins. They play a crucial role in various biological functions and are often used in research, pharmaceuticals, and health supplements. However, peptides are usually sold in a lyophilized (freeze-dried) form, which requires reconstitution before use.

Question 1

Do peptides degrade?

Accepted Answer

Peptide degradation factors, influenced by biological processes, temperature variations, and freeze-thaw cycles, can significantly impact the stability and effectiveness of peptide solutions, necessitating careful storage and handling procedures. 
Proteolytic enzymes within the biological system play a vital role in peptide degradation. Factors such as pH levels, presence of metal ions, and reactive oxygen species can accelerate the breakdown of peptides.
Temperature sensitivity further exacerbates this process, as both heat and cold can destabilize peptide molecules. Freeze-thaw cycles, common during storage and handling, can cause physical stress and lead to structural changes in peptides, affecting their bioactivity.
To mitigate these risks, it is essential to store peptides at recommended temperatures, utilize protective additives, and adhere to proper handling techniques to ensure optimal peptide stability and functionality.

Question 2

Shelf Life of Reconstituted Peptides?

Accepted Answer

Understanding the shelf life of reconstituted peptides and the factors that influence their stability under varying storage conditions is essential for maintaining the efficacy and quality of peptide solutions for research applications. 
Peptides, as delicate molecules, are susceptible to degradation over time due to factors like temperature, light exposure, and pH levels. Proper storage is crucial to extend the shelf life of reconstituted peptides.
Storing peptides in a cool, dark place away from direct sunlight can help prevent degradation. Reconstituted peptide solutions should be aliquoted and stored at recommended temperatures and concentrations to minimize potential damage. Implementing regular checks for signs of degradation and following manufacturer

Question 3

What is in the peptide vial?

Accepted Answer

The Peptide Vial: (GENERALLY YOU PURCHASE THESE FROM A TRUSTED ONLINE PEPTIDE VENDOR) 
This contains the peptide in a lyophilized (freeze-dried) powder form and Requires reconstitution with bacteriostatic water.(purchase from where you bought your peptides)(1)

Question 4

How do I solubilize my peptides?

Accepted Answer

Peptide solubility characteristics vary strongly from one peptide to another.  Residues such as Ala, Cys, Ile, Leu, Met, Phe, and Val will increase the chance of the peptide being hydrophobic and dissolving in aqueous solutions.  AnaSpec catalog peptides are tested for their solubility in certain solvents.  This information is located on every QC Datasheet that is shipped along with the peptide.  AnaSpec recommends that customers adhere to the below guideline for their custom peptides.

Solubility properties
Peptide solubility is highly dependent on the amino acid sequence. Hydrophobic peptides (high propensity of A, F, G, V, L, I, M, W, P) in nature, will require an organic solvent to dissolve. Acidic peptides (high propensity of D, E in the peptide sequence) require a basic aqueous buffer to dissolve, while basic peptides (high propensity of K, H, and R) require an acidic aqueous buffer to dissolve.

Selection of solvent
Taking into consideration the limitations of your assay, we recommend that the following guideline be used to determine the best solvent to dissolve your peptide 
 
Hydrophobic peptides
To reconstitute a hydrophobic peptide, add 100 µL of DMSO and sonicate until a homogenous solution forms. Next, add your buffer of choice to form a 1 mg/mL solution (a higher concentration of peptide will require a greater amount of DMSO).
 
Hydrophilic (acidic) peptide
To reconstitute an acidic peptide, add 100 µL of 1% NH4OH to 1 mg of the peptide and vortex.  After the formation of a clear solution, add your buffer of choice to form a 1 mg / mL solution. 
 
Hydrophilic (basic) peptide
To reconstitute a basic peptide, add distilled water to the 1 mg of peptide and vortex.   
 
Use and storage
Reconstituted peptides can be stored frozen at -20°C for a short time, but it is advisable to prepare multiple aliquots to avoid multiple freeze-thaw cycles. We recommend that all aliquoted solutions be lyophilized if the peptide is going to be stored for extended periods at -20 oC.
Peptides with a propensity to aggregate
For peptides that tend to aggregate due to the presence of multiple Cysteines, we recommend that these peptides be dissolved in degassed solutions and/or acidic conditions.
Additionally, some types of peptides have a propensity to form secondary and tertiary structures once dissolved.  We recommend that these peptides be first dissolved in solvents such as HFIP (hexafluoroisopropanol) and then evaporated using a stream of nitrogen.  The HFIP helps to break up the hydrogen bonding network that aids in forming the secondary and tertiary structures.

Question 5

What is the difference between net vs gross peptide weight?

Accepted Answer

The industry standard is to deliver peptides in a lyophilized form as a Gross weight. Gross peptide weight is the total weight of all components present in the lyophilized powder. This includes the peptide of interest, any peptide impurities, water, residual solvents, and counterion.
On the contrary Net peptide weight, calculated from the Net peptide content, is the weight of only the peptide component present in the lyophilized powder. This offers more accurate concentrations of your subsequent peptide solutions. Hence, we recommended net peptide weight amounts for Custom Peptide orders.

Net Peptide Content measurement is performed by amino acid analysis (AAA; limited accuracy but requires a low material amount) or elemental analysis (CHN; requires milligrams of peptide but is more accurate). Both methods yield a percentage value (ie., 75% net peptide content).
The Net Peptide Content depends on the amount of counterion present*, which is dependent on the peptide sequence. Essentially, the higher the peptide charge, the lower the peptide content (read more at FAQ: What is a Peptide Counterion?).
*Note: The element used to calculate net peptide content is based on the peptide counter-ion. This is done to ensure that the counter-ion does not contribute to the peptide content amount. For example, when TFA or acetate are the counter-ion, nitrogen is used because it is found in the peptide but not in TFA or acetate. Carbon or Nitrogen can be used used when chloride is the counter-ion for the same reason.
Net Peptide Weight calculations:
Net Peptide Quantity (total peptide) — method we use for net custom peptide orders.
Multiply the Net Peptide Content Percentage (decimal form) by the Peptide Gross Weight, to obtain the amount of Net Peptide Quantity (total peptide).
For example:
For 5 mg gross peptide/vial:
Peptide net content is 84%.
Exact amount of total peptide = 0.84*5 mg=4.2 mg total peptide
Net Peptide of Interest Quantity—most accurate — customer can use this method on their own to calculate peptide of interest.
Multiply Net Peptide Content Percentage by the Peptide Gross Weight, and the Peptide Purity (by HPLC), to calculate the exact amount of the peptide of interest you have. The net peptide percent (decimal form) is multiplied by peptide purity (decimal form).
For example:
For 5 mg gross peptide/vial with:
Peptide net content is 84%.
Exact amount of peptide of interest is = 0.84*0.96*5 mg=4.03 mg

Question 6

How long do peptides last in the fridge? How should peptides be stored?

Accepted Answer

For long-term storage, the peptide should be kept in solid form in the deep freezer at < -15 °C. If stored at room temperature some peptides containing methionine or cysteine may begin to degrade. Therefore, we recommend storing them at -20C as soon as possible after receiving the package. At -20 or -80, the peptides will remain potent for 6 months or years before beginning to degrade. For short-time storage, a refrigerator (+4 °C) will suffice. Peptides should be protected from intense sunlight. Peptides containing fluorophores should be kept in the dark.

Question 7

What may happen during the peptide storage?

Accepted Answer

Some typical degradation reactions or racemization may include the oxidation of Met, Trp, Tyr, or Cys. The deamidation of Asn, Gln, and the C-terminal amide may happen. The aspartimide may form.  There might be the cleavage of Asn-Pro.  The N-terminal Gln may form pyroglutamine.  The dimerization of Trp and Tyr may form.

Question 8

Why Reconstitute Peptides?

Accepted Answer