 |
What chemistry do you use to produce synthetic peptides?
|
 |
| Q. What chemistry do you use to produce synthetic peptides? |
|
A. PEPTRON: Fmoc solid-phase peptide synthesis is employed to build linear peptides.
Peptide is synthesized from its C-terminus by stepwise addition of amino acids.
Initially, the first Fmoc-amino acid is attached to an insoluble support resin via an acid labile linker. After deprotection of Fmoc by treatment with piperidine, the second Fmoc-amino acid is coupled utilizing a pre-activated species or in situ activation. After the desired peptide is synthesized, the resin bound peptide is deprotected and detached from the resin via TFA cleavage. |
 |
|
|
|
How do I store my synthetic peptides?
|
|
| Q. How do I store my synthetic peptides? |
|
A. PEPTRON: (Most) lyophilized peptides will be stable at room temperature for at least several months.
For long-term storage and maximum stability, lyophilized peptides should be stored at -20 (or preferably at -80). Repeated freeze-thaw cycles should be avoided.
Before use, let the container and its contents warm to room temperature to avoid absorbtion of water.
Peptide stability becomes much worse when in a solution, especially at the higher pH (pH>8). Since peptides in solution are unstable at temperatures even lower than -20, a peptide solution once prepared should be used as soon as possible. Please keep the peptide solution cold, and do not freeze again.
If peptide samples need to be frequently or periodically taken from the stock, it is recommended to make a series of aliquots from the stock, and freeze-thaw cycles should be avoided by freezing individual aliquots at -80. |
|
|
|
|
How do I dissolve water insoluble peptides?
|
|
| Q. How do I dissolve water insoluble peptides? |
|
A. PEPTRON: If the peptide is hardly soluble in aqueous solution, sonication may help break up any particles and increase the rate of dissolution.
For basic peptides 10%(or more) acetic acid is recommended whereas for acidic peptides aqueous ammonia or 10% ammonium bicarbonate solution is helpful. Peptides having poor solubility in aqueous solutions organic solvents have to be applied to get these peptides in solution. Use the minimal amount of organic solvent (DMSO is preferred, but DMF, isopropanol, methanol can also be used) to dissolve your peptide and slowly add water until the final concentration is reached. |
|
|
|
|
What is the purity of peptide?
|
|
| Q. What is the purity of peptide? |
|
| A. PEPTRON: Crude (desalting grade) peptide is desalted after synthesis and its purity is >50% by HPLC, whereas HPLC grade peptides is purified by HPLC to the purity (usually 90% or 95%) required by customer. |
|
|
|
|
What are the impurities?
|
|
| Q. What are the impurities? |
|
| A. PEPTRON: Most impurities are incorrectly synthesized peptides, such as fragments or deletion peptides, and incompletely deprotected peptides. |
|
|
|
|
What is the method of Fluorescein labeling?
(And, what is the difference between Fluorescein and FITC?)
|
|
| Q. What is the method of Fluorescein labeling? (And, what is the difference between Fluorescein and FITC?) |
|
A. PEPTRON: FITC (Fluorescein isothiocyanate) is activated precursor used for the Fluorescein labeling.
For the efficient N-terminal labeling, a seven-atom aminohexanoyl spacer (NH2-CH2-CH2-CH2-CH2-CH2-COOH) is inserted between the fluorophore (fluoroscein) and the N-terminus of the peptide.
This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated and making it more accessible to secondary detection reagents. |
|
|
|
|
Is C-terminal labeling of Biotin (or FITC) possible?
|
|
| Q. Is C-terminal labeling of Biotin (or FITC) possible? |
|
| A. PEPTRON: C-terminal labeling of Biotin (or FITC) is done by addition of a Lys residue at the C-terminus of a peptide, and Biotin (or FITC) is attached to the lysine side chain via amide bond. Lysine's positive charge is removed. |
|
|
|
|
What is the appropriate peptide length for antibody production?
|
|
| Q. What is the appropriate peptide length for antibody production? |
|
A. PEPTRON: Generally, 10~20 (mostly 15) residue peptide is recommended.
Longer peptide could have more epitopes, but could have more chance to form stable secondary structure which is not native form.
Shorter peptide is generally not a good unless there are valid reasons for it such as potential sequence homology with a related family member or other proteins. |
|
|
|
|
Conjugation method?
|
|
| Q. Conjugation method? |
|
A. PEPTRON: In order to generate immune response, peptides should be conjugated to bigger carrier proteins. You have a choice of BSA, ovalbumin, or KLH.
One of the advantage of KLH (keyhole limpet hemocyanin) is that it does not interfere with ELISA or Western Blot because it is not used for blocking reagent.
General conjugation method is maleimide method which couples the Cysteine residue of the peptide to the carrier protein.
For the peptide conjugation, one cysteine residue should be added to N- or C-terminus of the peptide in order to be linked to carrier protein.
Please select a less important terminus to be conjugated to protein.
if your peptide is N-terminal sequence of a protein, addition of Cysteine to C-terminus will be good, and vice versa.
If your peptide has internal cysteine residue, C-terminal conjugation via the carboxyl group by EDC method is used when there is no Glu, Asp residues, or N-terminal conjugation via amino group by Glutaraldehyde method is used when there is no Lys residue. |
|
|
|
|
What kind of terminal choice is appropriate?
|
|
| Q. What kind of terminal choice is appropriate? |
|
A. PEPTRON: If your peptide is internal sequence of a protein, terminal amidation (C-terminus) or acetylation (N-terminus) will be desirable in order to remove the charge and to imitate its natural structure (amide, CONH2).
In addition this modification makes the resulting peptide more stable towards enzymatic degradation resulting from exopeptidases. |
|
|
|
|
Which purity is recommended for my application?
|
|
| Q. Which purity is recommended for my application? |
|
A. PEPTRON: >75%: immunological applications and nonsensitive screening
>90%: SAR studies, bioassays
>95%: NMR, crystallization, bioassays, in-vivo studies |
|
|
|
|
How are the peptides shipped?
|
|
| Q. How are the peptides shipped? |
|
| A. PEPTRON: Peptides are provided lyophilized and as gross weight in small polypropylene vials (2 ml) with screw caps. |
|
|
|
|
Should I consider adding a Cysteine in peptides for making antibodies?
|
|
| Q. Should I consider adding a Cysteine in peptides for making antibodies? |
|
A. PEPTRON: Chemical conjugation using Cysteine offers a single point attachment provided there is just one Cys in the sequence (added or part of the native sequence). It is preferable to add Cys at the NH2 terminus if the peptide is internal or it represents the very C-terminus. This will keep the COOH free (non-conjugated) as it exists in native protein. For peptides representing the very NH2-terminal sequences, Cys should be added at the C-terminus of the peptide. For internal peptides, Cys can be added at either end but it is easier to synthesize peptides containing a NH2-terminal Cysteine. Cysteine can also be used to couple peptides to Sepharose for affinity purification of antibodies.
Amino or COOH-conjugation chemistries should be avoided as most peptides contains several NH2 and COOH groups available in a given peptide sequence resulting into multi-point attachment and peptide distortion. |
|
|
|
|
What is a MAP?
|
|
| Q. What is a MAP? |
|
| A. PEPTRON: MAPS or Multi-Antigenic Peptide is a branched peptide at which linear peptide chains are linked at their C-terminus via polylysine core, thereby increasing the size of whole molecule. This is done to eliminate the coupling of peptides to KLH. It seems that, however, conformation of peptides on MAP is less flexible, and antibodies obtained by MAP often recognize protein less often than by conventional KLH conjugation. In addition, there is no free peptide produced when making MAP, making it difficult to remove polylysine core directed antibodies. Purification of MAP by HPLC is difficult, and MAP is provided without mass verification due do its heterogeneity and large molecular size. |
|
|
|
|
Why does my KLH/Peptide solution appear cloudy?
|
|
| Q. Why does my KLH/Peptide solution appear cloudy? |
|
| A. PEPTRON: KLH or Keyhole Limpet Hemocyanin is a large (MW = 4*105 - 1*107) aggregating protein. Because of its size and structure, its solubility in water is often limited, causing a cloudy appearance. This does not affect immunogenicity and the turbid solution can be used for immunizations. |
|
|
|
