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Peptide Quick Tips

What impurities are in my peptide sample?

Common impurities found in custom peptide material can include any of the following:

  • Sequences with amino acid deletions
  • Incomplete peptide sequences (truncation)
  • Sequences containing incompletely deprotected amino acids
  • Sequences modified as a result of the cleavage process  (i.e. reattachment of protecting groups at other locations on the peptide)
  • Peptides that have undergone side reactions such as proline isomerization or isoapartimide formation, etc.
  • Trace amounts of residual DTT
  • Trace amounts of residual TFA
  • Trace amounts of Acetic acid

Peptides purified by reverse-phase chromatography (HPLC) will have the majority of these impurities removed.

Selecting the best purity level based upon application

Because peptides can be used in a variety of applications, purity requirements will  differ. Examples of common applications and the recommended  purity levels:

>70% Purity

>80% Purity

>95% Purity

Antigen for antibody production

Phosphorylation studies

In vitro and in vivo studies

ELISA standard

Non-quantitative enzyme-substrate studies

NMR studies

Peptide array production

Affinity purification

Mass spectrometry

Competitive elution chromatography

Protein electrophoresis applications

Quantitative assays

 

Difference between gross weight and net weight

Gross peptide weight is the weight of the peptide plus peptide impurities as well as extraneous components such as salts, counterions, residual solvents and water.  Unless requested, all peptides are supplied based upon gross weight.

Net peptide is typically determined by quantitative amino acid analysis (AAA), resulting in a known amount of full length product.  Net peptide content should not be confused with peptide purity.  Purity defines the percentage of the target peptide sequence in the peptide component of your sample.  AAA is available for an additional fee.

Estimating the peptide amount based upon final purity and gross weight

Peptide amount from gross weight is calculated using the following formula:

Gross Weight (in mgs) X Final Purity (from CofA) = Peptide amount
Example: Gross Weight (in mgs) = 5.1; Final Purity (from CofA) = 95% 5.1mgs X 0.95 = Estimated 4.845 mgs of full length peptide product.

Peptide classification

Use the following guidelines to determine if the peptide is acidic, basic, or neutral:

Step 1. Assign a value of –1 to each acidic residue (D, E and C-terminal COOH).
Step 2. Assign a value of +1 to each basic residue (K, R and the N-terminal NH2).
Step 3. Assign a value of +1 to each H residue at pH <6 or zero at pH >6.
Step 4. Add up the total number of charges of the peptide at pH 7 (all D, E, K, R, C-terminal COOH and N-terminal NH2) to calculate the overall net charge of the peptide.

Guidelines for peptide solubilization

Peptide Sequence Classification

Recommended Solvent*

Acidic (net negative charge)

0.1M Ammonium Bicarbonate

Basic (net positive charge)

25% Acetic acid

Neutral (net zero charge; >25% charged amino acids)

0.1M Ammonium Bicarbonate

Neutral (net zero charge; <25% charged amino acids)

Acetonitrile (ACN), DMSO or DMF

Hydrophobic peptide

Acetonitrile (ACN), DMSO or DMF

*Add recommended solvents in small amounts and slowly dilute (dropwise) with water to the desired concentration.

Other Considerations

Before trying stronger solvents, sonicate the peptide solution to confirm that the peptide is insoluble in the chosen solvent. Sonication enhances solubilization, breaking the solid peptide into smaller particles. If, after sonication, the solution has gelled, appears cloudy, or has visible particulates, the peptide has not dissolved completely but is suspended. At this point, a stronger solvent is necessary. If the peptide does not dissolve, lyophilize and remove the volatile buffer solution. Once the sample is dry, alternative solvents can be tested  on the same sample.

Guidelines for storage and handling

Helpful guidelines for proper storage and resuspension of your peptide for optimal performance in your assay. Learn more