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Transcript
Custom Peptide Technical Resource Amino Acid Table Amino Acid Name Alanine Arginine Asparagine Aspartic Acid Cysteine Glutamic Acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine 3-Letter Code Ala Arg Asn Asp Cys Glu Gln Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val 1-Letter Code A R N D C E Q G H I L K M F P S T W Y V Side Chain Polarity Nonpolar (hydrophobic) Polar (soluble) Polar Polar Polar Polar Polar Nonpolar Polar Nonpolar Nonpolar Polar Nonpolar Nonpolar, aromatic Nonpolar Polar Polar Nonpolar, aromatic Polar, aromatic Nonpolar Amino Acid Structures Side Chain Acidity/Basicity Neutral Basic (strongly) Neutral Acidic Neutral Acidic Neutral Neutral Basic (weakly) Neutral Neutral Basic Neutral Neutral Neutral Neutral Neutral Neutral Neutral Neutral Hydropathy Index 1.8 -4.5 3.5 -3.5 2.5 3.5 -3.5 0.4 -3.2 4.5 3.8 -3.9 1.9 2.8 -1.6 -0.8 -0.7 0.9 -1.3 4.2 Molecular Weight 89.09 174.20 132.12 133.10 121.16 147.13 146.15 75.07 155.16 131.17 131.17 146.19 149.21 165.19 115.13 105.09 119.12 204.23 181.19 117.15 Isoelectric Point 6.00 11.15 5.41 2.77 5.02 3.22 5.65 5.97 7.47 5.94 5.98 9.59 5.74 5.48 6.30 5.68 5.64 5.89 5.66 5.96 Peptide Design You need to consider the following items when design your peptide. Nonpolar, Aliphatic R Groups - - - H H - - CH3 O- H O H-N+-C-C CH OH CH2 CH2 O- - - - H-N+-C-C H 1. Peptide purity For antibody generation and testing, peptide purity > 70% is enough, however, for biological activity studies, peptide purity > 95% is required. Proline (Pro or P) - - - H-N+-C-C - O- H - - H Serine (Ser or S) H H O - - - - - - - - H-N+-C-C Alanine (Ala or A) H H O OH Glycine (Gly or G) H H O Polar, Uncharged R Groups CH2 H - - H - - H H CH2 O- - 3. Peptide length Peptide length governs the level of difficulty when synthesizing peptides. Peptide antigens are recommended to contain 10-15 residues to be used in antibody production. CH2 O- H C-NH2 CH2 O-C-NH2 O CH3 O H-N+-C-C H-N+-C-C CH2 O- - - - - Glutamine (Gln or G) O - - - - - - O S-CH2 H3C SH H - H - - H CH2 O- H Asparagine (Asn or N) H O H-N+-C-C CH OCH3 - - - - - - - Methionine (Met or M) H-N+-C-C - - - - H H H H-N -C-C + CH H3C CH3 - - H-N+-C-C 2. Peptide amino acid composition Amino acid composition governs every aspect of the peptide’s functionality. Hydrophobic and hydrophilic characteristics are key factors to consider. The peptide should incorporate antigenic amino acids sequence and avoid problematic amino acids such as cysteine, methionine, and tryptophan. Cysteine (Cys or C) Threonine (Thr or T) H H O CH2 O- H - - - - - - - - lsoleucine (Ile or I) H H O CH O CH2 CH3 O H-N+-C-C H CH OH3C CH3 H H H - - - - - - - H-N+-C-C Leucine (Leu or L) OH - - Valine (Val or V) H H O Positively Charged R Groups Aromatic R Groups H CH2 O - OH - - - - - - - Arginine (Arg or R) H O H CH2 O- H C N+H CH HC-N - H CH2 H3N+-CH2 H O H-N+-C-C H-N+-C-C CH2 OCH2 Tryptophan (Trp or W) H H O H - - H - - - - Histidine (His or H) O CH2 OCH2 + NH2 CH2 - CH2 O- H - H H-N -C-C + - - - - - H H-N+-C-C H - - - - - - O - - H - - - - - H H-N+-C-C Tyrosine (Tyr or Y) H H O - - - - Phenylalanine (Phe or F) - - - - - - Lysine (Lys or K) H2N-C- NH Negatively Charged R Groups H-N+-C-C H O CH2 O H H O H-N+-C-C H-N+-C-C H Glutamic acid (Glu or E) - - H - - - - - NH Aspartic acid (Asp or D) - - CH2 OC- CH - - - - H - 4. Peptide solubility You can design peptides of varied solubility by changing the frequency of occurrence of hydrophobic and hydrophilic amino acids. Hydrophobic amino acid content is suggested to be kept below 50% with at least one charged residue incorporated within every five amino acids. In addition, a single conservative replacement or addition of polar residues to the N- or C-terminus may also improve peptide solubility. CH2 O- H CH2 C- O O - O C- O 5. Secondary structure During peptide synthesis, beta sheet formation can cause incomplete dissolution of the growing peptide thus deletion sequences in the final product. Please avoid 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 (D), Glu (E), and the C-terminal -COOH. Assign a value of +1 to basic residues which include Arg (R), Lys (K), His (H), and the N-terminal -NH2. Calculate the overall charge of the entire peptide. a. Storage Lyophilized peptides should be refrigerated and stored away from bright light. For short-term storage ranging from one week to two months, lyophilized peptides may be stored at 4°C. For 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 peptides expose to moisture, therefore, the vial should be tightly capped at all times. This also requires careful experiment planning to minimize vial openings. 2. If the overall charge of the peptide is positive, try to dissolve the peptide in water first. If water fails, try 10%-30% acetic acid solution. If the peptide still does not dissolve, add TFA (< 50 μl) to solubilize the peptide and then dilute the peptide solution to the desired concentration. 3. If the overall charge of the peptide is negative, try to dissolve the peptide in water first. If water fails, add NH4OH (< 50 μl) and then dilute the peptide solution to the desired concentration. If the peptide contains Cys, then do not use basic solutions to dissolve it. Try the alternative method listed below. Do not store excessive peptides in solution because the shelf life of peptides in solution is very limited, much shorter than lyophilized peptides. This is especially true for peptides whose 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 re-lyophilize the remaining peptide solution. Another drawback is that peptides stored in solution are susceptible to bacterial degradation. If storage of peptide in solution is unavoidable, use sterile buffers at pH 5-6 and store aliquots at -20°C and pass the peptide solution through a 0.2 μM filter to rid the bacteria. b. Handling 4. Peptide whose overall charge is zero usually dissolves in organic solvent. First, try to add some acetonitrile, methanol, or isopropanol. For very hydrophobic peptides, try dissolving the peptide in a small amount of DMSO, and then dilute the solution with water to the desired concentration. For Cys-containing peptides, use DMF instead of DMSO. For peptides that tend to aggregate, add 6 M guanidine•HCl or 8 M urea, and then proceed with the necessary dilutions. When preparing peptides for use, please consult GenScript's following guidelines: • Warm the peptide vial to room temperature before opening. • Weigh out the desired quantity of peptide quickly. • Reseal the vial tightly. • Store the remaining peptide in a freezer, preferably below -20°C, under dry conditions. Avoid repeated freeze and thaw cycles for both lyophilized peptides and peptide solutions. If frequent and periodical sample takings from storage are unavoidable, GenScript recommends 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. Applications Recommended Purity • • • • • • • • • • • • • • • Immunograde Peptide Peptide purity >70% Biochemistry Grade Peptide Peptide purity >85% High Purity Grade Peptide Peptide purity >95%->98% Antigen for production and affinity purification of polyclonal antibodies ELISA tests Peptide array Epitope mapping Semi-quantitative enzyme-substrate studies Phosphorylation studies Peptide blocking studies by Western blotting In-vitro bioassays Cell attachment studies Quantitative assays NMR studies In-vitro bioassays In-vivo studies Clinical trials SAR studies 860 Centennial Ave., Piscataway, NJ 08854, USA Web: www.genscript.com Toll-Free: 1-877-436-7274 Fax: 1-732-210-0262 Tel: 1-732-885-9188 Email: [email protected]