Saporin

Manufactured by Merck Group

Sourced in United States

Saporin is a laboratory reagent produced by Merck Group. It is a ribosome-inactivating protein that functions to inhibit protein synthesis in target cells.

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Lab products found in correlation

14 protocols using saporin

Peptide-Saporin Complexes: Efficient Protein Transduction

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Formation of the peptide/saporin mixture: for a triplicate, 0.54 μg of saporin (from Saponaria officinalis seeds, Sigma) at 0.1 mg/mL in water was added to the desired amount of peptide diluted in 30 μL of PBS and the mixture was then gently mixed. Protein complexes were generated using increasing peptide/protein w/w ratios. After 30 min of incubation at room temperature, the mixture was diluted with serum-free culture medium to obtain a final volume of 0.225 mL.
Protein transduction: in total, 12,000 HCT116 cells were plated in 96-well plates one day before the experiment. Then, 75 µL of serum-free medium containing the peptide/protein complexes was transferred into each well of the triplicate. After an incubation time of 2 h 30 min at 37 °C the medium was removed and replaced with a fresh one containing serum. After 48 h of incubation, 20 µL/well of the MTS reagent was added. The cytotoxicity assay was then performed as described below.

Lointier M., Dussouillez C., Glattard E., Kichler A, & Bechinger B. (2021). Different Biological Activities of Histidine-Rich Peptides Are Favored by Variations in Their Design. Toxins, 13(5), 363.

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Saporin-FITC Conjugation Protocol

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Two hundred microgram of saporin (saporin from Saponaria officinalis seeds, Sigma-Aldrich) dissolved in H₂O (100 μl) was reacted with 2 equivalents of FITC (Sigma-Aldrich) dissolved in dimethyl sulfoxide (10 μl) and N,N-diisopropylethylamine (0.5 μl) at 30 °C for 2 h. For the removal of unreacted FITC, gel filtration on a Sephadex G-25 column (PD-10, GE Healthcare) was conducted prior to lyophilisation. The protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific Inc.).

Nakase I, & Futaki S. (2015). Combined treatment with a pH-sensitive fusogenic peptide and cationic lipids achieves enhanced cytosolic delivery of exosomes. Scientific Reports, 5, 10112.

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Saporin-Mediated RNA Modification Protocol

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Saporin treatment
and aniline cleavage were performed according to a previous report.^{3 (link)} Briefly, DNA oligos or in vitro-transcribed RNA
was incubated with the indicated amounts of Saporin (S9896, Sigma-Aldrich)
in 30 μL of reaction buffer [25 mM Tris (pH 7.5), 25 mM KCl,
and 5 mM MgCl₂] at 37 °C for 30 min or as long as
indicated. The reactions were quenched by adding SDS to a final concentration
of 2% from a 10% stock solution. After Saporin treatment, the oligos
were precipitated by addition of 0.1 volume of 3 M sodium acetate
and 3 volumes of 99% ethanol with centrifugation at 18000g and 4 °C for 15 min. The pellets were resuspended in 20 μL
of acetic acid/aniline (2.5 and 1 M) and incubated at 60 °C for
5 min. Aniline was removed by extraction with 500 μL of diethyl
ether and acetic acid by an additional round of ethanol precipitation.
Finally, the pellets were resuspended in 3 μL of water and 3
μL of 2× RNA loading buffer for PAGE analysis or purification.
For the confirmation experiment with RNA, the reaction was scaled
down using half of the amounts of all components.

Hauf S., Rotrattanadumrong R, & Yokobayashi Y. (2022). Analysis of the Sequence Preference of Saporin by Deep Sequencing. ACS Chemical Biology, 17(9), 2619-2630.

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Synthesis and Characterization of Novel Reagents

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Acryloyl chloride (CAS 814686), glycine (CAS 56-40-6), 4-aminobutanoic acid (CAS 56-12-2), 6-aminocaproic acid (CAS 60-32-2), 8-aminooctanoic acid (CAS 1002-57-9), 11-aminoundecanoic acid (CAS 2432-99-7), B7 (CAS 4687949), B8 (CAS 15625895), E1 (CAS 109-76-2), FITC-BSA, saporin from Salvia officinalis seeds, FITC-IgG from human serum, and Cas9-NLS were purchased from Sigma-Aldrich (St. Louis, MO). S4 (CAS 133251005) was purchased from Alfa Aesar (Tewksbury, MA).

Rui Y., Wilson D.R., Choi J., Varanasi M., Sanders K., Karlsson J., Lim M, & Green J.J. (2019). Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing. Science Advances, 5(12), eaay3255.

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Cytotoxicity Assay with Saporin or Cytochrome c

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Raw264.7-H2Kb cells (0.1 X 10⁶ per well in 96-well plates) were treated with or without 50 ng/mL Bafilomycin A1 (SelleckChem, US) as inhibitor, and then treated with toxin (either 25 ng/mL Saporin (Sigma, US) or 2.5 mg/mL Cytochrome c (Sigma, US)) in the presence of the indicated adjuvants for 5 h at 37°C. Then, media was removed, and cells were washed with PBS. Fresh media was added, and cells were incubated overnight. Supernatant LDH as cell death indication was analyzed by CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, US).

Lv X., Martin J., Hoover H., Joshi B., Wilkens M., Ullisch D.A., Leibold T., Juchum J.S., Revadkar S., Kalinovska B., Keith J., Truby A., Liu G., Sun E., Haserick J., DeGnore J., Conolly J., Hill A.V., Baldoni J., Kensil C., Levey D., Spencer A.J., Gorr G., Findeis M, & Tanne A. (2024). Chemical and biological characterization of vaccine adjuvant QS-21 produced via plant cell culture. iScience, 27(3), 109006.

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Dendrimer-Based Targeted Drug Delivery

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Generation 5 amine-terminated polyamidoamine dendrimer (G5-NH₂) with an ethylenediamine core was obtained from Dendritech Inc. (Midland, MI, USA). 4-(Bromomethyl)phenylboronic acid was purchased from Meryer Chemical Technology Co., Ltd. (Shanghai, China). Saporin and PASP sodium (2000–11000 Da) were purchased from Sigma-Aldrich (St Louis, MO). Cy5.5 NHS ester was purchased from Lumiprobe (USA). Fluorescein isothiocyanate (FITC) was bought from Macklin Biochemical Co., Ltd. (Shanghai, China). Methanol and dimethyl sulfoxide (DMSO) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sangon Biotech. (Shanghai, China). BCA assay kit were purchased from Beyotime (Jiangsu, China).

Yan Y., Zhou L., Sun Z., Song D, & Cheng Y. (2021). Targeted and intracellular delivery of protein therapeutics by a boronated polymer for the treatment of bone tumors. Bioactive Materials, 7, 333-340.

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Preparation of SAVA Conjugates

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SAVAs have been prepared as previously described (Antonucci et al., 2012 (link)). Briefly, 1 mg reduced rabbit anti-VGAT-C (131103, Synaptic Systems, Göttingen, Germany) was coupled to 2 mg of saporin (Sigma-Aldrich, Taufkirchen, Germany) with the bifunctional cross-linker sulfosuccinimidyl 6-[α-methyl-α-(pyridyldithio) toluamido]hexanoate (sulfo-LC-SMPT, Thermo Fisher Scientific, Schwerte, Germany). Free saporin was removed by affinity purification of SAVA using VGAT-C immunogen immobilized on sulfolink coupling resin (Thermo Fisher Scientific).

Reichel J.M., Nissel S., Rogel-Salazar G., Mederer A., Käfer K., Bedenk B.T., Martens H., Anders R., Grosche J., Michalski D., Härtig W, & Wotjak C.T. (2015). Distinct behavioral consequences of short-term and prolonged GABAergic depletion in prefrontal cortex and dorsal hippocampus. Frontiers in Behavioral Neuroscience, 8, 452.

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Fluorescent Saporin and EGF Conjugation

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Saporin (200 μg, Saporin from Saponaria officinalis seeds, Sigma-Aldrich) or EGF (200 μg) dissolved in H₂O (100 μl) was mixed with FITC (2 equivalents, Sigma-Aldrich) dissolved in dimethyl sulfoxide (10 μl) and N,N-diisopropylethylamine (0.5 μl) at 30 °C for 2 h. Gel filtration on a Sephadex G-25 column (PD-10, GE Healthcare) was conducted for the removal of unreacted FITC prior to lyophilisation. Concentration of the protein was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific Inc.).

Nakase I., Kobayashi N.B., Takatani-Nakase T, & Yoshida T. (2015). Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes. Scientific Reports, 5, 10300.

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Photodynamic therapy efficacy assessment

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MC28 cells were seeded out at a density of 3000 cells in 96 well plates overnight. Cells were then treated with 19 or 26 (50 nM) or saporin (Sigma-Aldrich Gillingham, UK) (20 nM) for 18 h separately. Another group of cells were co-incubated with saporin (20 nM) and 19 or 26. Cells were then washed twice with PBS and incubated for a further 4 h with fresh full medium. Irradiation was carried out for up to 5 min using a blue LumiSource® flatbed lamp with peak emission at 420 nm and 7 mW cm^–2 output (PCI Biotech, Oslo, Norway). Cell viability was determined by means of the MTT assay 72 h after the light exposure. Each experiment was carried out in triplicate.

Dondi R., Yaghini E., Tewari K.M., Wang L., Giuntini F., Loizidou M., MacRobert A.J, & Eggleston I.M. (2016). Flexible synthesis of cationic peptide–porphyrin derivatives for light-triggered drug delivery and photodynamic therapy †Electronic supplementary information (ESI) available: Copies of 1H and 13C spectra, HPLC traces, UV and fluorescence spectra. See DOI: 10.1039/c6ob02135b Click here for additional data file.. Organic & Biomolecular Chemistry, 14(48), 11488-11501.

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Saporin-Mediated Cytotoxicity Modulation

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HeLa cells were transferred into 96-well plates (1 × 10⁴ cells/well) and cultured for 24 h. After complete adhesion, HeLa cells were treated with 100 μl saporin (Sigma-Aldrich) in the presence or absence of 6 (20 μM) at 37 °C for 1 h. After incubation, the cells were washed twice with PBS and incubated in DMEM at 37 °C for 6 h (26 (link)). Cytotoxicity was then analyzed by MTT assay.
For the 4 °C experiments, HeLa cells were precultured at 4 °C for 30 min, then treated with 100 μl saporin (20 μg/ml) in the presence or absence of 6 (20 μM) at 4 °C for 1 h. After incubation, the cells were washed twice with PBS and incubated in DMEM at 37 °C for 6 h. Cytotoxicity was then analyzed by MTT assay.
For the endocytosis inhibitor experiments, the cells were preincubated in DMEM containing 100 μM EIPA, 10 μM NY, or 5 μg/ml CPZ for 30 min respectively. Then, HeLa cells were treated with 100 μl saporin (20 μg/ml) in the presence or absence of 6 (20 μM), as well as corresponding inhibitor, at 37 °C for 1 h. After incubation, the cells were washed twice with PBS and incubated in DMEM at 37 °C for 6 h. Cytotoxicity was then analyzed by MTT assay.

Chen X., Liu H., Li A., Ji S, & Fei H. (2021). Hydrophobicity-tuned anion responsiveness underlies endosomolytic cargo delivery mediated by amphipathic vehicle peptides. The Journal of Biological Chemistry, 297(6), 101364.

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