1.What synthesis methodologies do Crefu Peptides use for peptide synthesis?
RE: Crefu Peptides have developed different solution phase synthesis methodologies to meet customers' requirements. Usually, solid phase Fmoc chemistry is applied.
2.How do you ship peptides? What QC data will be provided?
RE: All peptides will be shipped by DHL or FedEx express as in lyophilized powder in individual well-labeled vials, at room temperature. With the exception of crude peptides, which will only be checked by MALDI-MASS spectrometry, all synthetic purified peptides comes with project report (CoA), MS data and HPLC data. data sheets will be provided that contains the key characteristics, such as peptide sequence, purity, quantity, modification, mass spectrum data and HPLC data.
3.What is the typical turn-around time for peptide synthesis? How long does it take to ship to me?
RE: Our typical turn-around time is 2-3 weeks for a standard peptide under 30 amino acids. The turn-around time varies depending on the peptide length, quantity, solubility and difficulty. Typically 3-5 days to reach researchers in other countries.
4.How long you can synthesize?
RE: Crefu Peptides has extensive experience in synthesizing long peptides, up to 130aa. Unlike many peptide suppliers who are only comfortable in making peptides under 30 or 40aa, Crefu Peptides has good experience in making peptides ranging from 40aa to 90aa. It is difficult to synthesize long peptides, especially 100aa or longer. If you plan to synthesize a sequence of 130aa or longer, please contact us for custom protein expression and purification service.
5.What if some problems come up during the synthesis or purification process?
RE: Each peptide has its specific characteristics. If some problems happen during the synthesis beyond our expectation,and we cannot deliver your peptide on time, we will inform you as soon as possible. By chance that we are not able to make the peptide, you will not be charged for any costs.
6.TFA salt form, Acetate or HCl salt form: Which form should I choose?
By default, peptides are synthesized in TFA salt. For cell or tissue culture related experiments, you should consider having peptides produced in acetate or HCl salt form at 98% or higher to avoid abnormal responses. Acetate or HCl salt form can be requested at additional cost.
7.What is the shelf life of a peptide?
RE: Peptides are stable for minimum one year if stored lyophilized in freezer. If peptides are dissolved the shelf life might be reduced. If peptides contain several reactive amino acids that can be oxidized like Trp, Met but especially Cys, the shelf life might also be reduced.
8.The peptide contains several cysteines and can therefore easily form disulfide bonds. How do I avoid this?
RE: If the peptide sequence contains several cysteines, or other reactive amino acids, which are easily oxidized, Crefu Peptides offers to deliver the peptide with traces of the strong reductant DTT. The peptide is only delivered with DTT if this is specifically permitted by the customer.
9.How do I store my synthetic peptides?
RE: Most lyophilized peptides will be stable at room temperature for 2-3 weeks. For long-term storage, you should store lyophilized peptides at -20°C. Repeated freeze-thaw cycles should be avoided. Allow to come to room temperature before opening. The shelf life of peptide solutions is limited; a peptide solution once prepared should be used as soon as possible.
10.What is the purity for the crude peptide and the desalted peptide? How do you purify the peptide? What are the impurities?
RE: For short peptides with normal sequences under 15aa, it is generally 40-60% by HPLC for crude grade; 50-70% by HPLC for desalted grade. The longer the peptide, the lower the purity for crude or desalted. Peptides are generally purified by HPLC using water and acetonitrile gradient. Most impurities are fragments or deletion peptides, incompletely de-protected peptides, and residual salt and water.
11.Explanation of Peptide Purity by HPLC, Total Peptide Content and Target Peptide Content.
RE: Crefu Peptides ships peptides according to the gross weight of the lyophilized powder. The lyophilized powder includes impurities such as fragments or deletion peptides, incompletely de-protected peptides, TFA and residual water. Peptide Purity is measured by HPLC. It is the amount of correct peptide relative to all analytes that absorb at ~214 nm (the peptide bond absorbs), most likely deletion, truncation or incompletely de-protected sequences, etc. Peptide purity by HPLC does not take into account water and salts that are usually present in the sample. Total Peptide Content is the percentage of total peptides present relative to everything present in the lyophilized peptide powder. Total peptide content is determined by nitrogen content analysis. Target Peptide Content in the lyophilized peptide powder thus can be concluded by the formula: Total Peptide Content X Peptide Purity by HPLC.
12.What purity levels are available and what's the best purity level for my application?
RE: Crefu Peptides offers different purity levels starting with crude, desalted, > 70% up to > 98%.
Please, refer to the following guideline, to select the most suitable grade of purity for your application:
Peptide purity >75%, preferably >85%
Immunological applications Antibody production
Non-sensitive screening
ELISA standard for measuring antibody titer
Peptide purity >90%
Non-quantitative enzyme-substrate studies
Phosphorylation assays
Non-quantitative peptide blocking studies
Affinity purification
Coating tissue culture plates for cell attachment
Peptide purity >95%
In-vitro bioassays SAR studies
Enzymology
Quantitative ligand-receptor interaction research
Quantitative blocking assays
Quantitative ELISA standards
Peptide Purity >98%
Active pharmaceutical ingredients
Clinical trials Crystallography
NMR
Sensitive Bioassay
13.What is the method of Fluorescein labelling?
RE: FITC (Fluorescein isothiocyanate) is the activated precursor used for the Fluorescein labelling. For the efficient Nterminal labelling, 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.
14.Is C-terminal labelling of Biotin (or FITC) possible?
RE: Yes. C-terminal labelling of Biotin (or FITC) is done by addition of a Lysine residue at the C-terminus of a peptide, and Biotin (or FITC) is attached to the Lysine side chain via an amide bond. Lysine's positive charge is removed.
15.What is the appropriate peptide length for antibody production?
RE: Generally, a 10-25 residue peptide is recommended. A longer peptide could have more epitopes, but could also have a greater chance of forming stable secondary structures which are not native forms. A shorter peptide (<10aa) is generally not good unless there are valid reasons for it, such as potential sequence homology with a related family member or other proteins.
16.What is a MAP?
RE: 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 typically recognize target protein less often than by conventional KLH conjugation. In addition, there is no free peptide produced when making MAPS, making it difficult to remove polylysine core directed antibodies. Purification of MAPS by HPLC is difficult, and MAPS is provided without mass verification due do its heterogeneity and large molecular size.
17.Why does my KLH-conjugated peptide solution appear cloudy?
RE: KLH or Keyhole Limpet Hemocyanin is a large aggregating protein (MW = 4x105 – 1x107). Because of its size and structure, its solubility in water is limited, causing a cloudy appearance. This shall not affect immunogenicity and the turbid solution can be used for immunizations.
18.Can you explain the M+Na and M+K mass peaks in MALDI spectra?
RE: It is very common to see Na (sodium) and K (potassium) adducts in the MALDI spectrum. The sodium and potassium comes from the water used in the peptide solvents. Even distilled and deionized water has trace amounts of sodium and potassium ions, which can never be entirely removed. These become ionized during the MALDI mass spec process and bind to the free carboxyl groups of the peptide. Because there is no water purification system that will remove every single sodium or potassium ion from water, seeing the sodium and potassium adducts at times is very common and unavoidable in MALDI mass spec. This is not an indication that the peptide is not pure, nor should it be confused with an incorrect molecular weight.
