The most stable way to store peptides for long periods of time is to keep them lyophilized, away from light and at low temperatures. However, when it comes time to use them, the work of reconstituting lyophilized peptides can be a headache.
Achieving the correct dissolution of the peptides is essential for the proper development of the assay, since otherwise peptide losses and / or inaccuracies may occur when calculating their concentration.
In this post we bring you some tips and guidelines that can help you reconstitute lyophilized peptides, and optimize their solubility .
Before proceeding to reconstitute lyophilized peptides, it is worth remembering some general considerations :
- As a previous step to reconstitution, it is always convenient to centrifuge the vials containing the lyophilized peptide so that the complete sample remains in the pellet and no material is lost.
- Before reconstitution, the peptide must be at room temperature .
- It is recommended to test the solubility in a certain medium with a small amount of peptide , until confirming that it dissolves correctly.
- The solvent of choice must be compatible with the experimental application and must not react or promote degradation of the peptide.
- In general terms, the solubility of a peptide will be determined by its net charge , therefore:
- Acid peptides can be resuspended in basic buffers.
- The basic peptides will be resuspended in acidic solutions.
- Hydrophobic and neutral peptides should be dissolved in small amounts of some organic solvent such as DMSO, methanol, acetic acid, isopropanol, etc.
- In order to improve solubility, complementary techniques such as sonication can be used , if necessary.
When choosing the optimal solvent to reconstitute lyophilized peptides, it is necessary to take into account the two fundamental factors that will influence its solubility : the length of the peptide and its net charge.
1.- PEPTIDE LENGTH
As a general rule, peptides of less than 5-6 amino acids are usually soluble in aqueous solution. It should be remembered that distilled and sterile water should always be used.
For larger peptides, the solubility will be defined by their net charge and hydrophobicity, as detailed in the second point.
2.- NET CHARGE OF THE PEPTIDE
To calculate the net charge of the peptide, the following procedure will be followed, and the obtained values will be added:
- A value of -1 will be assigned for each acidic residue (Asp, Glu or C-terminal -COOH) that contains the peptide.
- A value of +1 will be assigned for each basic residue (Arg, Lys, His or N-terminal -NH2) that contains the peptide.
Depending on the net charge value obtained, we will act as follows:
- If the net charge of the peptide is negative (acid peptide):
- First, attempts will be made to reconstitute the lyophilized peptide in water or PBS.
- If that first option fails, NH4OH will be added (except in cases where the peptide contains Cys residues).
- If the peptide still does not dissolve, an organic solvent such as DMSO will be added until it dissolves, and then it will be diluted to the desired concentration.
- If the net charge of the peptide is positive (basic peptide):
- First, an attempt will be made to reconstitute the lyophilized peptide in water.
- If that first option fails, an attempt will be made to dissolve the peptide in a 25% acetic acid solution.
- If the peptide still does not dissolve, an attempt will be made to solubilize in a small amount of DMSO and then dilute to the desired concentration.
- If the net charge of the peptide is equal to 0 (neutral peptide):
- First, attempts will be made to reconstitute the lyophilized peptide in an organic solvent.
- In cases where the peptide is highly hydrophobic, an attempt will be made to solubilize in a small amount of DMSO and then it will be diluted with water to achieve the desired concentration.
Although in many cases when reconstituting lyophilized peptides these can be dissolved without problem in aqueous solutions, it is important to know the characteristics of the amino acids that compose them to be able to predict the solubility in each case. We hope that this entry has cleared some doubts about it.