My peptide is 26 amino acids long (mol wt : around 2850 g/mol), and is known to be a cell-penetrating peptide, I want to track its localisation in cell by labelling it with FITC. I am thinking of labelling the N-terminus. There are 3 other lysine residues. How to block these? Could some one help and give me a detailed protocol on how to do this, remove the unlabelled peptide and dye molecules and how to confirm the labelling?
The labelling of peptides with FITC is usually done in hydrogen carbonate/carbonate buffers at pH 8.5-9. Alpha amino groups are deprotonated at this pH-value, but not epsilon amino groups. They have a higher pKa-value (pKα-NH3+ : 8,90, pKε-NH3+ : 10,28). This means if you label a peptide with FITC in a buffer with pH-value of 8.5-9, the epsilon amino groups of lysine should not react with FITC.
So, you could try at first to label the peptide without protection of the side groups of the lysine residues.
The removal of non-reacted FITC and peptide residues from the reaction mixture is usually done by gelfiltration on G10 or G25 Sephadex columns in water followed by collection of the reaction products (detection by UV-spectroscopy and thin-layer chromatography) and removal of water by lyophilisation.
The labelling can be confirmed by fluorescence spectroscopy. In the frst step, you can check whether you have got one or several yellowish coloured and fluorescent reaction products by thin-layer chromatography on silicagel.
It may be that you get reaction with some of the epsilon amino groups of the lysine residues, too. To avoid this, you should perform the labelling at a pH-value not higher than 9.0. If you know the N-terminus amino acid of your peptide, you should look for its pKa value.
In the worst case, you have to use a protective group for the epsilon lysine residues. Such a protective group could be the 4-methyltrityl group (Mtt). This Mtt group is selectively removed with 5 % TFA, 5 % triisopropylsilane (TIS) in dichloromethane (DCM) for 15 min.
References:
Maeda, H; Kawauchi, H, New method for the determination of the N- terminus of a peptide chain with fluorescein isothiocyanate, Biochemical and Biophysical Research Communications (1968), 31(2), 188-92
Rinderknecht, H, Experientia XVI (9), (1960), 430-431
Kawauchi, H; Tuzimura, K; Maeda, H; Ishida, N, J. Biochemistry 66 (6), (1969), 783-789
Muramoto, K; Meguro, H; Tuzimura, K, Agric. Biol. Chem. 41 (10), (1977), 2059-2062
Burtnick L D, Modification of actin with fluorescin isothiocyanate, Biochimica et biophysica acta (1984), 791(1), 57-62
Dear Dr. Schaefer,
Thank you so much for the detailed answer to my question, Very informative indeed for a beginner like me. Really appreciate this help!
Maybe you solved this already but I find that carboxyflourescin is cheaper and easier to work with and good enough to follow a peptide inside of cells. You just couple it at the end of the synthesis to the n-terminal in the same way that you would couple an amino acid monomer. And all of your lysine residues are still protected.
It can be coupled using DIC, or H/TBTU + DIPEA, I usually go overnight at room temperature or just 5 minutes a 70C. Just remember to wash the resin with 20% piperidine before cleaving the peptide, you will notice that the color of the resin intensifies. Then just purify normally using HPLC the fraction containing the labelled peptide will be visibly yellow. Of course this requires you to make the entire peptide yourself.
I am not sure if the answer is still relevant.
Although I am not from the chemistry field, but I have also used FITC tagged peptide for my study in the following papers:
https://www.researchgate.net/publication/319647058_Efficient_intracellular_delivery_of_biomacromolecules_employing_clusters_of_zinc_oxide_nanowires
https://www.researchgate.net/publication/310838911_Generation_of_protective_immunity_against_Orientia_tsutsugamushi_infection_by_immunization_with_a_zinc_oxide_nanoparticle_combined_with_ScaA_antigen?_iepl%5BviewId%5D=psbdKr3XHWaSQdARDSCAzcBJnlSs1Bf59J9C&_iepl%5Bcontexts%5D%5B0%5D=prfhpi&_iepl%5Bdata%5D%5BstandardItemCount%5D=3&_iepl%5Bdata%5D%5BuserSelectedItemCount%5D=0&_iepl%5Bdata%5D%5BtopHighlightCount%5D=2&_iepl%5Bdata%5D%5BstandardItemIndex%5D=2&_iepl%5Bdata%5D%5BstandardItem2of3%5D=1&_iepl%5BtargetEntityId%5D=PB%3A310838911&_iepl%5BinteractionType%5D=publicationTitle
I guess ordering from a company can be a best option.
We have asked a company to send us peptide with FITC tagged, and it didn’t cost very much.
I hope it helps
Henrik Helmfors that was a detailed reply, thanks. May I ask if you know why the colour of the resin intensifies after using Piperidine after Fluorescein coupling? and why do we need to use piperidine if there is no Fmoc group for deprotection? Thank you
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