Top 5 reasons your peptide-based assays fail

You planned your peptide assay carefully from start to finish, so why did your experiment fail? Learn about the intrinsic features of custom peptides that can cause high experimental variability or total assay failure – and how to prevent future assay failures.

5. Biological Contamination

Why it can ruin your assays
Are your immunostimulation assays generating erratic data? Endotoxin contamination may be the culprit. Peptides contaminated with lipopolysaccharides, which are widely called endotoxins (major components of the cell walls of gram-negative bacteria), can lead to variations in immunological assays, as they can stimulate a plethora of unwanted immune reactions in B cells, and macrophages, as well as stimulate T cell expansion. Even at low concentrations, endotoxins can stimulate the production of interleukins, tumor necrosis factors, prostaglandins, and platelet activating factors.

What You Should Do

Endotoxins can be introduced into peptide preparations during the peptide synthesis process or whenever your peptide vial is open in the lab. In addition, lab glassware and other containers can be contaminated with endotoxins even after being autoclaved. To limit endotoxin contamination open peptide vials only in a sterile hood, and use certified endotoxin free centrifuge tubes, and glassware.

4. TFA Counter-ion Contamination

Why it can ruin your assays

Observing erratic cell growth or death in your cellular assays? The cause could be trifluoroacetate counter-ions. Trifluoroacetic acid (TFA) is a strong acid, which is commonly used to cleave synthesized peptides from solid-phase resins and is also used to improve HPLC performance in the peptide purification step. During lyophilization of the purified peptide, free TFA is removed, but residual TFA remains in the peptide preparation in anionic form. Thus, peptides are usually delivered as TFA salts.

Residual TFA has been found to interfere with cellular assays, inhibiting cellular proliferation in some instances (Cornish et al.), and increasing cell viability in others (Ma et al.). It has also been found to be an unintended allosteric modulator of the glycine receptor (GlyR, (Tipps et al). Peptide-based assays in which TFA acts as a cell growth stimulator or inhibitor can introduce experimental variability, as well as false positive or reduced signals.

In addition, TFA has been found to interfere in infrared (IR) absorption spectroscopy since it has a strong absorbance band at 1673 cm-1 (Andrushchenko VV et al.). This band overlaps or obscures the amide I absorbance band of peptides, complicating peptide secondary structure determination experiments.

Finally, TFA can reduce the pH of a peptide preparation, and thus may alter the pH of subsequent assays.

What you should do

TFA removal service is available from GenScript Peptide Synthesis Services for custom peptides that may be used in TFA-sensitive cellular or enzymatic assays. GenScript uses anion exchange or reversed phase HPLC to exchange TFA counter-ions for acetate or HCl counter-ions, which have been shown to be more compatible than TFA in biological assays.

3. Improper Storage Conditions

Why it can ruin your assays

Improper peptide storage can lead to peptide degradation. Peptides should always be stored at -20°C and away from light. Whenever possible, peptides should remain in lyophilized form until just prior to their dissolution for assay. If you must store your peptides in solution form, use sterile buffers for the solubilization process and/or use a 0.2 µm filter to remove potential microbial contamination. Freeze-thaw cycles should also be avoided for both lyophilized and solution form peptides, as frequent freezing and thawing results in degradation. Degradation can result in the generation of new peptide species and a loss in peptide activity over time, which can skew data and thwart experimental reproducibility.

What you should do

Use our solubility guidelines to determine the best way to dissolve your peptide. For maximum dissolution, request a peptide solubility test from GenScript. The test report will tell you the best buffer and pH in which to maximally dissolve your peptide. Use our online quotation system to get a quote for your peptide and request a solubility test.

What you should do

Upon receiving your custom peptide, immediately aliquot the lyophilized powder according to your daily experimental needs (or request free aliquoting when you request a quote using our instant online quotation system). Store the aliquots at -20°C, and dissolve a single aliquot immediately prior to use.

2. Poor Peptide Solubility

Why it can ruin your assays

Having trouble dissolving your peptide? Not only is dissolving a hydrophobic peptide a pain, but precipitation of hydrophobic peptides can lead to assay variability. In addition, dissolving your peptide in the wrong buffer can ruin your custom peptide.

What You Should Do

Use our solubility guidelines to determine the best way to dissolve your peptide. For maximum dissolution, request a peptide solubility test when you use our online quotation system to request a quote for your custom peptide. The test report will tell you the best buffer and pH in which to maximally dissolve your peptide.

1. Oxidation

Why it can ruin your assays

Noticing a change in your peptide activity over time? Your peptide could be rapidly oxidizing. Peptides containing Cys, Trp, or Met are at great risk for oxidation. Oxidation of a peptide containing one or more of these residues can result in the generation of new peptidic species, such as peptides with oxidized side chain groups, peptide fragments resulting from backbone damage, and dimerized or aggregated peptides. Peptides that require reduced forms of Cys, Trp, or Met for activity may sub-perform in assays, resulting in decreased activity or no activity. A reduction in peptide activity may be observed over time resulting in a lack of assay reproducibility and increases in experimental data variability.

What You Should Do

To avoid the effects of peptide oxidation, store peptides in vials flushed with argon gas and tightly sealed. Make aliquots of lyophilized peptides according to daily experimental needs and limit opening and closing of peptide vials. You may also choose to dissolve peptides in buffers that have been flushed with argon or nitrogen gas to remove air, as well as flush assay buffers with argon or nitrogen gas. For extremely oxidation sensitive peptides, perform assays inside an anaerobic chamber to eliminate the effects of oxidation on your peptide-based assays.

GenScript ensures that your custom peptides arrive unoxidized and ready for assay. Each custom peptide is packaged using our ArgonShield™ packaging technology and peptide aliquoting is free (for up to 5 vials, then just $2 per vial).

Bonus: Improper peptide concentration calculation

Do your assays rely on knowledge of the exact concentration of your custom peptide? Are you experiencing batch-to-batch variability between peptide orders? Your peptide concentration calculations could be incorrect. First you must understand the difference between net peptide content, total peptide content, and peptide purity.

Peptide purity: When ordering custom peptides you select a peptide purity. This purity (e.g. 95%) represents the percentage of your peptide in a pool of peptidic impurities, which includes your sequence, as well as deletion sequences. Deletion sequences are partial sequences of your custom peptide that occur as a result of sequence truncation or amino acid deletion during the synthesis process.

Net peptide content: Expressed as a percentage, total peptide content consists of all peptidic material including your peptide sequence plus deletion sequences.

Total peptide content: Total peptide content is the total weight of the lyophilized peptide powder delivered to you. This includes all peptidic material (both your peptide sequence and deletion sequences) and other substances such as water, counterions, and salts.

Calculation of net peptide content can be estimated using the following equation (with the assumption that water and trace amounts of salts are negligible):

(mw of target peptide)

(mw of target peptide + # of TFA counter-ions*114)

where 114 is the MW of the TFA counterion

However, the only way to truly know the exact amount of your peptide is to request an Amino Acid Analysis Test, in which your custom peptide is hydrolyzed into individual amino acids, which are separated and quantified upon elution.


Andrushchenko VV et al. Optimization of the hydrochloric acid concentration used for trifluoroacetate removal from synthetic peptides (2007) J Pept Sci. 13:37-43.

Cornish J et al.Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes. (1999) American Journal of Physiology - Endocrinology and Metabolism 277: E779-E783.

Ma TG et al. Effects of trifluoroacetic acid, a halothane metabolite, on C6 glioma cells. J Toxicol Environ Health.(1990) 3:147-158.

Tipps ME, Iyer SV, Mihic SJ Trifluoroacetate is an allosteric modulator with selective actions at the glycine receptor. (2012) Neuropharmacology 63: 368-373.