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Common FAQ

Custom peptide synthesis FAQ

Peptide name, sequence lot number
Peptide molecular formula
Theoretical molecular mass
Molecular mass by mass spectrometry
Purity by HPLC
Appearance and solubility test

With very few exceptions, peptides in Qyaobio are synthesized by chemical methods. There are three major synthesis methods of peptides: including solid phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS), hybrid phase peptide synthesis (HPPS).

The peptide purity requirement is determine by the final applications.

Crude: Non-sensitive screening assays.
>70%: Non-sensitive screening assays, exploratory or relative MS assays.
>80%: ELISPOT assays, polyclonal antibody production, Non quantitative binding assays.
>85%: Immunological applications, polyclonal antibody production and non-sensitive screening.
>90%: SAR studies, receptor-ligand assays, bioassays.
>95%: In vitro bioassays such as ELISA, enzymology, Quantitative Mass Spectrometry SRM/MRM assays, monoclonal antibodies, quantitative receptor-ligated assays, enzyme kinetics, cell/tissue culture, biological activity.
>98%: Structural studies such as crystallography, NMR, X-ray or sensitive bioassays.

As most peptides contain the basic functionalities in sequences, such as the guanidino group of Arg, the ε-amino group of Lys, the imidazole moiety of His, the free N-terminus. All these basic moieties can form salt with acids. Except specific mentions, our peptides are provided as trifluoroacetate salts. Other salt forms are generated by an additional ion-exchange step, As the peptide will react to trifluoroacetic acid during the cleavage from the carrier resin.

Acidic peptides can form salts with bases, includes peptides with a large proportion of Asp, Glu, phospho, or sulfotyrosine. However, long peptides do not form stoichiometiric salts with acids or bases like low molecules.

As all research peptides are synthesized in TFA salt form. For cell-based assays or animal researches, we should remove TFA salt by switching it to HCl or acetate salt form with TFA<1%, in order to avoid the abnormal responses. Furthermore, higher purity>98% will get the optimal results depending on the budget.

In our normal peptide synthesis, Fmoc and Boc chemistry can both produce excellent peptides.

In hydrophobic peptide synthesis with high aggregation susceptibility, Boc chemistry provides a few advantages.

Removing Boc group in acid conditions will protonate the exposed amine terminal of peptides, this reduces the hydrogen bonding participation ,and increases its coupling availability.
Neutralization of activated amino acid will produce higher yields and purity peptides in difficult sequences.

Fmoc chemistry is more suitable for synthesis of larger peptides and small proteins with protected peptide fragments. Special resins are available for cleavage under mild conditions, in order to leave all other protecting groups intact.

The common method of solubility testing is based on charge determination. For small peptides up to 5 amino-acids, distilled water is the first option. Refer to the following guideline for other synthetic peptides.

Attribute -1 to every acidic residue(Asp, Glu) and the terminal carboxylic acid. Then assign -1 to each basic residues (Arg, Lys, His) and the terminal amine. Sum up both values to determine the overall charge of synthetic peptides.
Once the overall charge value is positive, try the distilled water at first. Then acidify the solution with the acetic acid (10 to 30%), when the peptides don’t dissolve. Add TFA if acetic acid does not allow peptide dilution to sufficient concentration.
If the overall charge value is negative, and peptides don’t contain cysteine, try to dissolve peptides in water. Then add ammonium hydroxide to achieve required concentration, once the peptides don’t dissolve.
In case the overall charge is zero, organic solvents like methanol, ethanol, isopropanol or acetonitrile, can be applied to dissolve peptides. According final application, the small amount of DMSO diluted with water can be employed. Furthermore, there are specific peptides are sensitive to oxidation, which contain cysteine, methionine or tryptophan residues, replace DMSO by DMF in these case.

Qyaobio synthesize peptides routinely between 20 and 90 amino acids. Although, we have extensive experience in peptide manufacturing from 40 AA to 100 AA. It is more difficult to synthesize peptide longer than 90 amino acids with high purity. In addition, Qyaobio is able to synthesize peptides up to 160 AA.

Chemically synthesized peptides have a free amine at the N-terminal and free acid at the C-terminal by default. We recommend the modifications for intra-cellular, in-vivo and in-vitro assays and researches. The modifications can increase the metabolic stability of peptides, and resistance to enzymatic degradation by exopeptidases, aminopeptidases, and synthetases.

Most lyophilized peptides are stable at room temperature for 2–3 weeks. Temperature at -20℃ is applied for long-time storage of lyophilized peptides. Furthermore, repeated freeze-thaw cycles must be avoided, and allow reaching room temperature before opening. The shelf life of peptide solutions is limited, it should be used as soon as possible once prepared.

There are several factors affect the peptide synthesis and purification, including amino acid succession, overall composition, peptide length, hydrophobic stretches.

Highly hydrophobic peptides are difficult to purify and solublize in aqueous solutions. These peptides normally involve residues like Leu, Val, Ile, Try, Phe and Met. Based on experience, we normally recommend one charged residue for every five amino acids.
Peptides with multiple Cys, Met or Try residues are difficult to achieve high purity, as these residues are prone to oxidation or hydrogen bonding. Normally, these peptides will result in slight delay, in reason of repeated synthesis and purification attempts.
Several Ser or Asp residues in one row will give rise to lower purity of peptides. Multiple Pro in sequence will create cis-trans isomerisation, this results in low apparent purity.
Peptides with the N-terminal Asn or Gln are difficult to achieve high quality, we recommend to remove, add or substitute with another amino acid.

Normally, the 10-25 residue peptides are recommended for antibody production. The longer peptides have more epitopes, but this also results in higher chance of forming in-native secondary structures with high stability. In addition, the shorter peptides less than 10 AA are not good enough unless valid reasons, such as potential sequence homology with the related protein family members.

The HPLC date refers to the information of peptide purity, it provides the percentage of target peptide in peptide material only, NOT the entire lyophilized powder.

The net peptides content refers to the percentage of peptide material in relation to the entire lyophilized powder. The typical net peptide content is 60-90%. Once for more accuracy, the net peptide content is achieved by Amino Acid Analysis (AAA), or Elemental Analysis (EA).

Generally, Qyaobio provides different types of cyclic reactions in peptides synthesis. In according, we have different analysis techniques to confirm the cyclizations.

Peptide cyclization of end to end, end to side, side to end are normally confirmed by both molecular shifts in Maldi-Tof mass spectrum and retention time alternative in analytical HPLC spectrum.

The dissulfide bridge cyclization of peptides are confirmed by HPLC before and after the cyclization step, the shift in retention time of the analytical HPLC spectrum can confirms the cyclic peptides.

For hydrocarbon-stapled peptides with alkene, the staple is confirmed by both the shift in molecular mass in mass spectrum, and retention time changes in analytic HPLC spectrum.

Common FAQ

Custom Antibody Production FAQ

Yes, we can provide them all. For antigen design, our peptide chemists can provide you with antigenicity analysis services and recommend the most immunogenic sequences with high probability of exposure. In terms of antigen production, we have extensive production experience, whether it is a gene, peptide or protein, etc., your antigen can be produced in house through synthetic or recombinant expression systems.

If you are immunizing with your own submitted antigens, the requirements vary based on the kind of protocol. For protein antigens, we recommend sending a minimum of 1-2 mg of antigen for rabbit projects and 5 mg for goat projects, with a purity >85% and the concentration needs to be at least 0.5 mg/ml. In the case of a peptide antigen, we expect 20 mg peptide (>85% purity).

Yes, our data sheets list species and applications for our validated antibodies.

There are many factors that influence whether an antibody attaches to another species, therefore even if the sequence alignment is excellent, we cannot ensure the antibody will work in an untested species.

Antibodies are only stable at room temperature for a few days, so we recommend that you store them at -20°C. Additionally, to avoid repeated freezing and thawing, you should also aliquot the antibody according to the experimental usage, with each aliquot should not be less than 10 μL. Because of evaporation of the liquid and adsorption to the container surface, too few aliquots affect antibody concentration.

Yes. The relevant proteins excised after staining should be mixed with a small amount of buffer to prevent the gel from drying out. Since protein concentration cannot be determined once the sample is received, you need to quantify the total protein amount as accurately as possible. Sometimes denatured proteins do not adhere well to ELISA microtiter plates, thus you may also wish to provide about 200 µg of protein in a non-gel state.

we will combine the peptide with KLH as a carrier protein. The KLH-peptides can be used with any peptide from any location within the protein.

The time it takes to obtain antibodies varies with different antibodies. Approximate time from start to finish for peptide antibodies is 16-24 weeks. Protein antibodies vary depending on expression system and production difficulty. The development of hybridoma takes 4 to 6 months.

Because of the serum antibody concentration and the anti-immunogen antibodies’ affinity, the yields from affinity purification can differ significantly. For every 100 mL of sera, 2–20 mg of purified anti-immunogen antibodies can be expected.

Crude ELISA titers are expressed as the inverse of serum dilution. A powerful, specific immune response is typically indicated by titers ranging from 1:50,000 to 1:100,000. For example, as shown by a titer of 1:50 000, when the antibody is attached to the solid phase at a dilution of 1:50,000, it can effectively detect the antigen.

Common FAQ

Custom Protein Production FAQ

Qyaobio can provide the following four expression systems: E. coli, yeast, insect and mammalian. We also offer a wide range of production and purification services, or offer other test services according to your requirements, such as tag removal, endotoxin removal, activity assessment, sequence identification, and QC testing, etc.

Our skilled protein team will go to great lengths to purify almost any protein. But for the highest chance of success, we recommend that target proteins have a molecular weight of less than 100kDa and avoid those with a great deal of transmembrane domains. Furthermore, protein expression will be inhibited by any signal peptide, which may be attached to the N-terminus of the protein in the recombinant system, therefore we also propose removal them.

Yes, we can make an enormous amount of protein in a single batch using E. coli expression systems. When accepting large scale projects, we usually begin with small-scale trial expression and purification. Then we send about 5 mg of the purified protein to you for testing. We can expand production, once it meets your requirements.

Our protein purity, as assessed by SDS-PAGE, is greater than 85%. Protein concentration is measured using the following three methods typically: Bradford, BCA, and A280 method. The Bradford approach is the most well-known and commonly used of these.

To address this issue, we recommend the following steps: (1) Codon optimization should be done if it has not already been performed; (2) The transmembrane region and signal peptide region was removed; (3) Truncated domain of the protein was expressed attemptly; (4) Try to increase expression levels by different tags; (5) We may suggest you to conside other expression systems (e.g. insect cell systems).

Post-translational modifications are generally responsible for the differences of products produced by different expression systems. Therefore, the application of purified protein and the source of your protein (bacterial or animal) decide which expression system to choose. If post-translational modification of the protein is not required, it is recommended to use E. coli protein expression system, which has low cost and high success rate. Otherwise, we will suggest yeast, insect cell, or mammalian cell expression systems based on your budget and specific requirements. Please ask us for a quote to determine the appropriate expression system for your target protein.

We offer codon optimization and gene synthesis services, so we can start with is the sequence information of your FASTA or NCBI entry. We can also use the construct you offer if your cDNA already exists in an expression vector. However, if you would like us to begin your plasmids or viruses, please send them to us.

Commonly used tags include His, GST, MBP, SUMO, Flag and combination tags (His-GST), etc.

Among them, His-tag is the most common one used for purification. Depending on the nature of the protein and the specific requirements of customer, our expert staff will suggest the appropriate tags for each type of protein. The final product will be tagless protein, which we can provide by cleaving the tags.

We provide free SDS-PAGE and Western blotting for all projects. You can order additional QC results with HPLC, MS, and N-terminal sequencing for modest additional fees.

We deliver in liquid form. To avoid multiple freezing and thawing, proteins are aliquoted into individual vials and store at -80°C or liquid nitrogen for long-term storage.