Strategies for the Synthesis of Labeled Peptides
Labeled peptides are frequently used by researchers for binding studies, to determine substrate specificity, and for receptor cross-linking studies. Many researchers would like to synthesize biotin, FITC, nanoparticle, or drug-labeled peptides. It is suggested that a new strategy, using Rink amide 4-methylbenzhydrylamine resin coupled with Fmoc-Lys(Dde)-OH, be used. The major advantage of this approach is that other labels such as FITC, dansyl groups, methyl coumarin, and potentially fluorophores and quenchers used for fluorescence resonance energy transfer (FRET) can be directly incorporated into peptides.
How much protein antigen do I need to send?
We recommend sending 4-6 mg and 90% purity of the antigen for a rabbit project. We recommend 0.5 mg/ml as a minimum protein concentration and welcome higher concentrations.
Does LifeTein guarantee antibody titer?
LifeTein guarantees at least a 1:32,000 antibody titer against peptide sequences that we synthesize/conjugate. Titers are confirmed via ELISA tests.
Peptide synthesis protocol
Take peptide Biotin-GLHSKKKLLHEKI for example.
Fmoc solid phase synthesis method was used for peptide production. Peptides were synthesized from the C-terminus to the N-terminus in this method. In peptide synthesis, an amino-protected amino acid Fmoc-Ile-OH was bound to a solid phase material (wang resin), forming a covalent bond between the carbonyl group and the resin (Fmoc-Ile-Wang resin). It was swollen by DCM, then the amino group fmoc was deprotected by piperidine, washed by DMF, and reacted with the carbonyl group of the next amino-protected amino acid Fmoc-Lys(Boc)-OH, with the help of HBTU in half an hour. The Kaiser test was taken to detect free ammonia. This cycle was repeated to form the desired peptide chain. For the last step of Biotin conjugation, fmoc group of the N terminus leu was deprotected and reacted with D-Biotin.
After all reactions were completed, the synthesized peptide was cleaved from the resin. The reagent K was TFA/TIS/water (95%/2.5%/2.5%). MS was performed to detect the M.W. The purification was under reverse-HPLC, C18 column, and 220nm wavelength preparation conditions.
Do I retain rights to the antibody and peptides produced by LifeTein?
You will receive all peptides and antibodies and retain rights to these materials. Your peptide sequence and protein information remain confidential and we will never release details of any potential, current, or past projects to any outside parties.
How to solubilize my synthetic peptides?
Please refer to this FAQ for details: http://lifetein.com/handling_and_storage_of_synthetic_peptides.html#q2.
If the peptides are still cloudy, or turbid, you may have reached the limit of solubility. When the peptides are insoluble in the buffer, please try to sonicate, centrifuge, and lyophilize the peptide. Make sure to break the lyophilized lumps into a fine powder. Then try a small volume of a good agent 8M Urea, NMP, DMF, or DMSO to dissolve the peptide. Then dilute with water or your desired buffer. For peptides with Arg or LYs, you make try to lower the pH to 6 because the protonated amino acids will help solubility.
Sonication and the following solvents may help for difficult peptides:
1) Begin with 100 % acetonitrile then diluted with water until 50%
2) Begin with 100% DMSO then diluted with water until 30 %
3) Dissolve it with 8M Urea
4) Dissolve it with 6 or 8 M Guanidine hydrochloride
5) 6M GuHCL, 0.05% TFA, pH2,
6) 100% TFA
7) 40% AcOH, 30%ACN, 30% water