It is always disappointing for the researcher if their experiments fail after carefully planning their peptide assays. It should be known that there are intrinsic features of custom peptides, which may not just cause huge experimental variability, but also total assay failure.
Here, we have a look at some of the reasons for assay failures, and what can be done to prevent failures in the future.
If you discover that your immunostimulation assays are offering erratic data, then endotoxin contamination is usually the number one culprit. When the peptides get contaminated with lipopolysaccharides, they can result in variations in immunology assays, and this can then lead to a plethora of unwanted reactions in macrophages and B cells.
To prevent biological contamination of the assays, the peptide vials should be opened only on a sterile hood. Additionally, concerning handling, certified endotoxin-free centrifuge tubes and glassware should be used.
Improper storage conditions
Improper storage conditions can also lead to peptide degeneration, ultimately leading to allay failures. It is recommended that peptides be stored at temperatures below -200C, and away from contact with direct light. If possible, peptides should be kept in lyophilized form until they are ready to be dissolved to be used for allays. If the peptides have to be stored in solution form, then sterile buffers must be used throughout the solubilization process, to stop any threats of microbial contamination.
Poor Peptide Solubility
Allay failures can also result in the following problems with respect to dissolving the peptide. It is not always easy to dissolve a hydrophobic peptide, and the precipitation of hydrophobic peptides usually tends to lead to assay variability. Additionally, using wrong buffers for the dissolution can ruin the custom peptides.
To avoid solubility problems, adhere to the solubility guidelines so that you come up with the best method to dissolve peptides. For maximum dissolution, be sure to request a solubility test from the vendor when buying custom peptides. From the test report, you can tell the best buffer and pH to use for optimal results.