N-terminus FITC labeling of peptides on solid support: the truth behind the spacer

Jullian, Magali; Hernandez, Anaïs; Maurras, Amélie; Puget, Karine; Amblard, Muriel; Martinez, Jean; Subra, Gilles

doi:10.1016/j.tetlet.2008.10.141

Cited by 90 publications

(77 citation statements)

References 14 publications

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“…We also devised an approach for staining intracellular proteins with fluorescein-5-isothiocyanate (FITC), a fluorochrome that covalently binds with proteins in vitro (Jullian et al, 2009). The specificity of RNA staining with AO and of protein staining with FITC was verified in mouse somatic fibroblasts and oocytes by their treatment with RNase A or proteinase K before cell exposure to the dyes.…”

Section: Introductionmentioning

confidence: 99%

Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA

Shishova

Lavrentyeva

Dobrucki

et al. 2015

Developmental Biology

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It is well known that fully-grown mammalian oocytes, rather than typical nucleoli, contain prominent but structurally homogenous bodies called "nucleolus-like bodies" (NLBs). NLBs accumulate a vast amount of material, but their biochemical composition and functions remain uncertain. To clarify the composition of the NLB material in mouse GV oocytes, we devised an assay to detect internal oocyte proteins with fluorescein-5-isothiocyanate (FITC) and applied the fluorescent RNA-binding dye acridine orange to examine whether NLBs contain RNA. Our results unequivocally show that, similarly to typical nucleoli, proteins and RNA are major constituents of transcriptionally active (or non-surrounded) NLBs as well as of transcriptionally silent (or surrounded) NLBs. We also show, by exposing fixed oocytes to a mild proteinase K treatment, that the NLB mass in oocytes of both types contains nucleolar proteins that are involved in all major steps of ribosome biogenesis, including rDNA transcription (UBF), early rRNA processing (fibrillarin), and late rRNA processing (NPM1/nucleophosmin/B23, nucleolin/C23), but none of the nuclear proteins tested, including SC35, NOBOX, topoisomerase II beta, HP1α, and H3. The ribosomal RPL26 protein was detected within the NLBs of NSN-type oocytes but is virtually absent from NLBs of SN-type oocytes. Taking into account that the major class of nucleolar RNA is ribosomal RNA (rRNA), we applied fluorescence in situ hybridization with oligonucleotide probes targeting 18S and 28S rRNAs. The results show that, in contrast to active nucleoli, NLBs of fully-grown oocytes are impoverished for the rRNAs, which is consistent with the absence of transcribed ribosomal genes in the NLB mass. Overall, the results of this study suggest that NLBs of fully-grown mammalian oocytes serve for storing major nucleolar proteins but not rRNA.

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Section: Introductionmentioning

confidence: 99%

Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA

Shishova

Lavrentyeva

Dobrucki

et al. 2015

Developmental Biology

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“…In this case, a b-alanine residue was introduced between the FITC chromophore group and the CCK8 N terminus to avoid fluoresceine elimination by the Edman degradation mechanism. 32 The assembly of pure and mixed aggregates was achieved providing energy to the system by sonication and extrusion procedures. In the case of mixed system, the amount of the two amphiphilic compounds, (C18) 2 DOTA and (C18) 2 L5CCK8, was selected to have a 90:10 molar ratio.…”

mentioning

confidence: 99%

Peptide‐labeled supramolecular aggregates as selective doxorubicin carriers for delivery to tumor cells

et al. 2011

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New liposomal aggregates, prepared by combining together, in a 90:10 molar ratio, two amphiphilic monomers, one containing two hydrocarbon chains in the hydrophobic region and the anionic DOTA chelating agent as hydrophilic moiety, and the other containing the same hydrophobic moiety and the CCK8 peptide, are described. The liposomal aggregates because of the presence of the specific moiety, constituted by the CCK8 peptide, which selectively recognizes CCK receptors on tumor cells are used as drug carriers with the aim to deliver into tumor cells the appropriate antitumor drug. The drug loading content and the releasing properties of the liposomal aggregates are studied by the use of the cytotoxic doxorubicin as drug model. The doxorubicin loading content determination reveals that above 95% of the total drug was uptaken with a corresponding drug/lipid w/w ratio of 0.134. The cellular uptake of the targeted liposomal doxorubicin with respect to the self-assembled, nonspecific, liposomal doxorubicin is evaluated using flow cytometry assays. The doxorubicin cell content for two types of cell systems, namely, A431 and HuVEC cells, for peptide derivatized liposomes was 70- and 8-fold higher, respectively, than for nontargeted liposomes, indicating that the bioactive CCK8 peptide is able to enhance the doxorubicin uptake into the carcinoma cells in vitro. The cytotoxicity effect of liposomal doxorubicin on A431 cells has been assessed by MTT assays: in presence of drug amounts ranged between 250 and 1000 ng/ml, incubation with peptide derivatized liposomes showed significantly lower cell survival compared with nontargeted liposomes.

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“…The Ahx spacer is critical at this point, since direct labeling of L-Lys by FITC may result in peptide degradation after cleavage from resin (60). Finally, all peptides were cleaved from resin and HPLC purified.…”

Section: Synthesis Of Kla Peptides With Lys-tpp At Either Nor C-terminimentioning

confidence: 99%

Multiple Triphenylphosphonium Cations as a Platform for the Delivery of a Pro-Apoptotic Peptide

et al. 2011

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N-terminus FITC labeling of peptides on solid support: the truth behind the spacer

Cited by 90 publications

References 14 publications

Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA

Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA

Peptide‐labeled supramolecular aggregates as selective doxorubicin carriers for delivery to tumor cells

Multiple Triphenylphosphonium Cations as a Platform for the Delivery of a Pro-Apoptotic Peptide

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