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TGpeptide proposes a range of different purity levels to help you make the right choice for your application. Crude peptides are not recommended for biological assays. Crude peptides may contain large amounts of non-peptide impurities such as residual solvents, scavengers from cleavage, TFA and other truncated peptides. TFA cannot be totally removed. Peptides with purity >70% are always used for generating or testing antibodis. Peptides with purity level >85% are usually used in enzymology or biological activity studies. Peptides with purity >95% are excellent for quantitative analysis. We also provide purity peptides with purity >98% in large quantities with commercial applications for our industrial customers. TGpeptide recommends the following levels of peptide purity for various projects:
Functional screening
Crude peptide
Peptide arrays
Antigens for antibody production
Competitive elution chromatography
ELISA standards for measuring antisera titers
70%and>75%
Western blotting studies (non-quantitative)
Enzyme-substrate studies (non-quantitative)
Peptide blocking studies (non-quantitative)
Affinity purification
Phosphorylation assays
Protein electrophoresis applications and immunocytochemistry
Purity >80%
Purity >85%
Purity >90%
ELISA standards and RIA protocols (quantitative) Purity >95%
Receptor-ligand interaction studies (quantitative)
In vitro bioassays and in vivo studies
Enzyme studies and blocking assays (quantitative)
NMR studies
Mass spectrometry
Other quantitative assays
95%
SAR Studies
Clinical trials
APIs (Active Pharmaceutical Ingredients)
Commercial products
X-ray crystallography studies
Other sensitive experiments: enzyme-substrate studies, receptor-ligand interaction studies,
blocking and competition assays
98%
Shipping the peptides: Peptides are shipped at room temperature, avoiding lights, and are highly stable at lyophilized form. Peptides should not be stored in solution even at the condition of -80℃.
Opening the package: Equilibrate the peptides to room temperature in a desiccator before opening and weighing.
Do not dissolve and lyophilize repeatedly the sequences that contain cysteine, methionine, tryptophan, asparagine, glutamine and N-terminal glutamic acid because they will have a shorter shelf life than other peptides.
Weigh out your required quantity of peptide rapidly and store all unused peptide at -20°C or below.
Solubility of peptide is mainly determined by its polarity. Acidic peptides can be dissolved in basic buffers, basic peptides can be dissolved in acidic solutions. Hydrophobic and neutral peptides who contain many hydrophobic or polar uncharged amino acids can be dissolved in small amounts of organic solvent such as DMSO, acetic acid, DMF, acetonitrile, propanol, methanol, or isopropanol, then diluted peptide using water carefully.
DMSO should not be used with peptides containing Met or free Cys because it might oxidize -SH group on the side-chain.
1，
First, assign a value of −1 to each acidic residue (Asp [D], Glu [E], and the C-terminal –COOH). Next, assign a value of +1 to each basic residue (Arg [R], Lys [K], His [H], and the N-terminal -NH2), then calculate the overall charge of the peptide.2，
The peptide is basic if the overall charge of the peptide is positive; The peptide is acidic if the overall charge of the peptide is negative; The peptide is neutral if the overall charge of the peptide is 0.3，
For basic peptide, try to dissolve the peptide in distilled water if possible. If it fails to dissolve in water, then try to dissolve the peptide in a small amount of 10–25% acetic acid. If this fails, add TFA (10–50 µl) to solubilize the peptide, and then dilute it to your desired concentration.4，
For acidic peptide, dissolve it in PBS (pH 7.4). If fails, add a small amount of basic solvent such as 0.1 M ammonium bicarbonate to dissolve the peptide, and then add water to the desired concentration. Peptides containing free cysteines should be dissolved in degassed acidic buffers because -SH group can be oxidized rapidly to disulfides if pH >7.5，
For neutral peptide, you can dissolve it in organic solvents. For very hydrophobic peptides, try to dissolve the peptide in a small amount of DMSO, and then dilute the solution with water to your 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.6，
If none of the methods in step 2 works, try the Trifluoroethanol (TFE). It was shown that TFE can induce and stabilize α-helices and induce β-turns, β-hairpins and also β-strands. Furthermore, a mixture of trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP) and trichloromethane (TCM) or dichloromethane (DCM) was found to be very powerful for dissolving peptides.Different experiments require different fluorescent dyes. Basically, experiments are divided in to two parts, which are vivo experiments and vitro experiments:
For vivo experiments, we should choose fluorescent dyes with excitation wavelengths between 650-900nm such as ICG、Cy5.5 and Nile Blue because they have better ability to pass the tissues and they get less disturbed from the background.
For vitro experiments, we usually use fluorescent dyes with excitation wavelengths between 400-600nm such as AMC、FITC and TAMRA.
Degradation of amino bond: Asn/Gln will be degraded to Asp/Glu in De Amino Bond reaction. In structure of Asn-Gly-, amino bond will be degraded more easily.
There are two reasons why peptide solution is easily to be oxidized. First one is that there are peroxide pollution in the solution; And the second one is the peptide itself is easily to self-oxide. Among all the amino residues, peptides containing Met、Cys and His、Trp、Tyr are very easily to be oxidized.
Hydrolyzation: Amino bond in peptide is easily to be hydrolyzed especially the amino bond consisting of Asp, Asp-Pro and Asp-Gly for example.
Form wrong disulfide bridge: If Cys in peptide forms wrong disulfide bridge, tertiary structure of peptide will be changed and cause the peptide inactivated.
α-Racemization: Except Gly, α-carbon atom of all amino acids is chiral, and they are easily to be racemized under basic conditions, Asp is easily to be racemized most.
β- Racemization: β- racemization means the racemization of group on βcarbon atom. Side group of Cys、Ser、Thr、Phe、Tyr is easily to be racemized at basic conditions.
Our peptides are synthesized using Solid Phase Peptide Synthesis(SPPS) technology based on Fmoc or t-Boc chemistry to protect the alpha amino group.
SPPS reaction are carried out on substrates covalently attached to a polymeric resin. Solid-phase synthesis can be better than the traditional synthesis because the overall reaction takes place much more quickly, the process can be automated with robots, and synthetic intermediates do not need to be isolated because reagents are washed away during each step.
The deprotection agent (piperidine for Fmoc, TFA for Boc) frees the alpha amino group in preparation for coupling the next amino acid in the sequence.
This reveals a new N-terminal amine to which the next amino acid may be activated by one of the several reagents, forming a peptide bond. When the synthesis is complete, peptides are cleaved from the resin and deprotected.
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