Custom Peptide Technical Resource

Amino Acid Table

Amino Acid 3-Letter 1-Letter Side Chain Side Chain Hydropathy Molecular Isoelectric Name Code Code Polarity Acidity/Basicity Index Weight Point Ala A Nonpolar (hydrophobic) Neutral 1.8 89.09 6.00 Arg R Polar (soluble) Basic (strongly) -4.5 174.20 11.15 Asn N Polar Neutral 3.5 132.12 5.41 Asp D Polar Acidic -3.5 133.10 2.77 Cys C Polar Neutral 2.5 121.16 5.02 Glu E Polar Acidic 3.5 147.13 3.22 Gln Q Polar Neutral -3.5 146.15 5.65 Gly G Nonpolar Neutral 0.4 75.07 5.97 His H Polar Basic (weakly) -3.2 155.16 7.47 Ile I Nonpolar Neutral 4.5 131.17 5.94 Leu L Nonpolar Neutral 3.8 131.17 5.98 Lys K Polar Basic -3.9 146.19 9.59 Met M Nonpolar Neutral 1.9 149.21 5.74 Phe F Nonpolar, aromatic Neutral 2.8 165.19 5.48 Pro P Nonpolar Neutral -1.6 115.13 6.30 Ser S Polar Neutral -0.8 105.09 5.68 Thr T Polar Neutral -0.7 119.12 5.64 Trp W Nonpolar, aromatic Neutral 0.9 204.23 5.89 Tyr Y Polar, aromatic Neutral -1.3 181.19 5.66 Val V Nonpolar Neutral 4.2 117.15 5.96

Amino Acid Structures Peptide Design

You need to consider the following items when design your peptide. Nonpolar, Aliphatic R Groups Polar, Uncharged R Groups 1. Peptide purity Glycine (Gly or G) Alanine (Ala or A) Serine (Ser or S) Proline (Pro or P) H H O H H O H H O H H O For antibody generation and testing, peptide purity > 70% is enough, however, for

－ － － － － － － － － －N+－－C －N+－－C －N+－－C － － － H C H C H C H－N+－－C C － － － － － － biological activity studies, peptide purity > 95% is required. - - -

－

HHO HCHO H CH O － - － 3 OH CH CH O － H 2 2

CH2 Valine (Val or V) Leucine (Leu or L) 2. Peptide amino acid composition H H O H H O Threonine (Thr or T) Cysteine (Cys or C) － － － － － － + H H H－N －－C C H－N+－－C C O H H O Amino acid composition governs every aspect of the peptide’s functionality.

－ － － － － － － + － － － HCHO- O- H－N －－C C + H CH2 H－N －C－C － －

－ -

－ － Hydrophobic and hydrophilic characteristics are key factors to consider. The peptide

H C CH H CH O - 3 3 CH OH H CH O － CH 2 3 － H3C CH3 SH should incorporate antigenic amino acids sequence and avoid problematic amino

lsoleucine (Ile or I) Methionine (Met or M) Asparagine (Asn or N) Glutamine (Gln or G) acids such as cysteine, methionine, and tryptophan. H H O H H O H H O H H O

－ － － － － － － － － －N+－－C － － － + + H C H－N+－－C C H－N －－C C H－N －－C C － － － - － － － － － - - H CH O - O O O H CH2 H CH2 3. Peptide length H CH2 CH CH － 2 3 －NH － C 2 CH2

SCH2 － H C － Peptide length governs the level of difficulty when synthesizing peptides. Peptide － 3 O － － CH O C NH2 3 antigens are recommended to contain 10-15 residues to be used in antibody Positively Charged R Groups Aromatic R Groups production. Lysine (Lys or K) Histidine (His or H) Arginine (Arg or R) Phenylalanine (Phe or F) Tyrosine (Tyr or Y) H H O H H O H H O － － － － － － － － － H H O H H O H－N+－－C C H－N+－C－C H－N+－C－C 4. Peptide solubility

－ － －

－ － － － － + － － － － H－N+－－C C H－N －－C C HCHO- O- O- 2 H CH2 H CH2

－ － You can design peptides of varied solubility by changing the frequency of occurrence － － － - O- － － H CH O H CH2 + 2 CH2 C N H CH2 － －

－ CH － of hydrophobic and hydrophilic amino acids. Hydrophobic amino acid content is CH HC －N － + CH 2 NH2 2 － －

－

－ H H N+－CH H N－C －NH OH 3 2 2 suggested to be kept below 50% with at least one charged residue incorporated within Tryptophan (Trp or W) every five amino acids. In addition, a single or addition of H H O Negatively Charged R Groups

－ － － H－N+－－C C polar residues to the N- or C-terminus may also improve peptide solubility. － － O- Aspartic acid (Asp or D) Glutamic acid (Glu or E) H CH2 － C －CH H H O H H O － NH － － － － － － H－N+－－C C H－N+－－C C 5. Secondary structure － － O- － － O- HCH2 H CH2 －

－ During peptide synthesis, beta sheet formation can cause incomplete dissolution of C － CH2 -

O O － C － the growing peptide thus sequences in the final product. Please avoid - O O multiple or adjacent residues of Val, Ile, Tyr, Phe, Trp, Leu, Gln, or Thr to prevent beta sheet formation. If the above recommendation cannot be implemented practically, conservative replacement may help by reinserting a Gly or Pro at every third residue or replacing Gln with Asp, or Thr with Ser. Peptide Solubilization Storage & Handling

1. Assign a value of -1 to acidic residues which include Asp a. Storage (D), Glu (E), and the C-terminal -COOH. Assign a value of +1 to basic residues which include Arg (R), Lys (K), Lyophilized peptides should be refrigerated and stored away from bright light. For short-term

His (H), and the N-terminal -NH2. Calculate the overall storage ranging from one week to two months, lyophilized peptides may be stored at 4°C. For charge of the entire peptide. long-term storage, lyophilized peptide should be stored at -20°C in deep freezer. Even though this condition keeps most peptides stable for several years, peptides' long term stability is at risk when 2. If the overall charge of the peptide is positive, try to peptides expose to moisture, therefore, the vial should be tightly capped at all times. This also dissolve the peptide in water first. If water fails, try requires careful experiment planning to minimize vial openings. 10%-30% acetic acid solution. If the peptide still does not dissolve, add TFA (< 50 μl) to solubilize the peptide and Do not store excessive peptides in solution because the shelf life of peptides in solution is very then dilute the peptide solution to the desired limited, much shorter than lyophilized peptides. This is especially true for peptides whose concentration. sequences contain cysteine, methionine, tryptophan, asparagine, glutamine, or N-terminal glutamic acid. It is necessary to aliquot the amount of peptide you need for the day and 3. If the overall charge of the peptide is negative, try to re-lyophilize the remaining peptide solution. Another drawback is that peptides stored in solution dissolve the peptide in water first. If water fails, add are susceptible to bacterial degradation. If storage of peptide in solution is unavoidable, use sterile NH4OH (< 50 μl) and then dilute the peptide solution to buffers at pH 5-6 and store aliquots at -20°C and pass the peptide solution through a 0.2 μM filter the desired concentration. If the peptide contains Cys, to rid the bacteria. then do not use basic solutions to dissolve it. Try the alternative method listed below. b. Handling

4. Peptide whose overall charge is zero usually dissolves in When preparing peptides for use, please consult GenScript's following guidelines: organic solvent. First, try to add some acetonitrile, • Warm the peptide vial to room temperature before opening. methanol, or isopropanol. For very hydrophobic peptides, • Weigh out the desired quantity of peptide quickly. try dissolving the peptide in a small amount of DMSO, • Reseal the vial tightly. and then dilute the solution with water to the desired • Store the remaining peptide in a freezer, preferably below -20°C, under dry conditions. concentration. For Cys-containing peptides, use DMF instead of DMSO. For peptides that tend to aggregate, Avoid repeated freeze and thaw cycles for both lyophilized peptides and peptide solutions. If add 6 M guanidine•HCl or 8 M urea, and then proceed frequent and periodical sample takings from storage are unavoidable, GenScript recommends with the necessary dilutions. making a series of aliquots in advance. Due to the fact that peptides whose sequences containing cysteine, methionine, or tryptophan are prone to air oxidation. It is recommended that air be purged from the peptide storage vial and replaced with inert gases such as argon or nitrogen for storage.

Applications

Peptides with purity greater than 70% are always used for generating or testing antibodies. Peptides with purity level greater than 85% are usually used in enzymology or biological activity studies. Peptides with purity greater than 95% are excellent for quantitative analysis.

Recommended Purity Applications

• Antigen for production and affinity purification of polyclonal antibodies Immunograde Peptide Peptide purity >70% • ELISA tests • Peptide array • Epitope mapping • Semi-quantitative enzyme-substrate studies Biochemistry Grade Peptide • Phosphorylation studies Peptide purity >85% • Peptide blocking studies by Western blotting • In-vitro bioassays • Cell attachment studies • Quantitative assays • NMR studies High Purity Grade Peptide • In-vitro bioassays Peptide purity >95%->98% • In-vivo studies • Clinical trials • SAR studies

860 Centennial Ave., Piscataway, NJ 08854, USA Web: www.genscript.com Toll-Free: 1-877-436-7274 Tel: 1-732-885-9188 Fax: 1-732-210-0262 Email: [email protected]