Digitonin

Manufactured by Thermo Fisher Scientific

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Digitonin is a natural detergent derived from the plant Digitalis purpurea. It is commonly used in biochemical research applications as a tool for cell membrane permeabilization and the extraction of proteins and other biomolecules from cells.

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Digitonin solution (4 citations) Digitonin/PBS (2 citations)

61 protocols using digitonin

Immunofluorescence Labeling of NG Neurons

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To label the isolated NG neurons a previously described indirect immunofluorescence technique was utilized (Blanke et al., 2019) . NG neurons (n=3 rats, 3 coverslips per animal) were plated on coverslips coated in concanavalin a and incubated overnight in MEM, as described above.
The cells were then fixed with fresh 4% paraformaldehyde and subsequently blocked with a solution containing 10% normal donkey serum (NDS, Cat. no. 017-000-121, Jackson Laboratories, Bar Harbor, ME)/ 1% bovine serum albumin (BSA, Cat. no. NC9871802, Fisher Scientific) in 1X PBS with 250 μg/mL digitonin (Cat. no. D14, Sigma-Aldrich). The neurons were then incubated for 3 hr with primary antibody (Ca V 2.2: 1:1000, Alomone Labs Cat# ACC-002, RRID: AB_2039766) in 3% NDS/1% BSA/ 0.05% sodium azide (Cat. no. 190380050, Thermo Fisher) diluted in 1X PBS with 250 μg/mL digitonin. Cells were washed three times with 1X PBS and then incubated for 30 min under dark conditions with the secondary antibody (Donkey antirabbit 1:2000, RRID:AB_2535792, Invitrogen) in 3% NDS/1% BSA in 1X PBS with 250 μg/mL digitonin. Finally, coverslips were again washed three times with 1X PBS and mounted using Aqua-poly/mount (Cat. no. 18606, Polysciences, Warrington, Pa) . In a separate set of control experiments, the primary or secondary antibodies were omitted during incubation to verify antibody specificity.

Goudsward H.J., Ruiz-Velasco V., Stella S.L., Willing L.B, & Holmes G.M. (2024). Coexpressed δ-, μ-, and κ-Opioid Receptors Modulate Voltage-Gated Ca²⁺ Channels in Gastric-Projecting Vagal Afferent Neurons. Molecular pharmacology, 105(3).

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Blue Native PAGE for Mitochondrial Protein Analysis

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BN-PAGE was performed as previously described [75 (link), 76 (link)]. Mitochondria (150 μg) were solubilized in 50 μl of NativePAGE Sample Buffer (4X; Invitrogen, catalog # BN20032) containing digitonin (Invitrogen, catalog # BN2006) at a digitonin/protein ratio of 4 g/g, on ice for 20 min. Solubilized proteins were supplemented with Coomassie brilliant blue G-250 (Invitrogen, catalog # BN2004) at a ratio of digitonin/Coomassie dye of 4:1. Samples were separated on 3–12% NativePAGE Bis-Tris gels (Invitrogen, catalog # BN2012BX10) and run with dark blue cathode buffer at 30 V for 1 h. The cathode buffer was then replaced with light blue buffer and the gels were run for 20 h at 30 V at 4°C. Dark blue cathode buffer was prepared by adding 50 mL of 20X NativePAGE Running Buffer (Invitrogen, catalog # BN2001) and 50 mL of 20X NativePAGE Cathode Buffer Additive (Invitrogen, catalog # BN2002) in 900 mL of deionized H₂O. Light blue cathode buffer was prepared by adding 50 mL of 20X NativePAGE Running Buffer and 5 mL of 20X NativePAGE Cathode Buffer Additive in 945 mL of deionized H₂O.

Oliva C.R., Ali M.Y., Flor S, & Griguer C.E. (2022). COX4-1 promotes mitochondrial supercomplex assembly and limits reactive oxide species production in radioresistant GBM. Cell Stress, 6(4), 45-60.

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Characterization of ATP Synthase Oligomerization

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ATP synthase oligomerization was analyzed by one-dimensional blue native (BN-) and clear native (CN-) polyacrylamide gel electrophoresis (PAGE). Protein complexes were extracted from INS-1E cells or INS-1E cell mitochondria using digitonin (Thermo Fisher; 2.5:1, w:w, protein:digitonin ratio). The obtained lysates were separated on bis-Tris 3%–13% gels. Following BN/CN PAGE proteins were transferred by wet electroblotting onto PVDF membranes. ATP synthase and DAPIT were immunodetected with monoclonal antibodies against the ATP synthase F₁α-subunit (Abcam, ab110273) and DAPIT/USMG5 (Abcam, ab108225) after acidic membrane-stripping.

Leguina-Ruzzi A., Vodičková A., Holendová B., Pavluch V., Tauber J., Engstová H., Dlasková A, & Ježek P. (2020). Glucose-Induced Expression of DAPIT in Pancreatic β-Cells. Biomolecules, 10(7), 1026.

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Purification of PS1 Complexes from HEK293F Cells

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HEK293F cells were harvested at a density of 3 million cells/ml, homogenized in 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 5 mM MgCl₂, 5 mM CaCl₂ at 4°C. Cells were lysed in the same buffer containing protease inhibitor cocktail (Roche), 1% (w/v) digitonin (Calbiochem) for 1 hr, and centrifuged at 100,000 × g for 1.5 hr. PS1 complexes were captured on a rabbit IgG-agarose column (Sigma-Aldrich), washed with 100 column volumes of buffer containing 0.04% (w/v) digitonin, cleaved with AcTEV protease (Invitrogen), and eluted with buffer containing 0.04% digitonin. The eluate was purified by Strep-Tactin chromatography (IBA GmbH); eluted in 0.04% digitonin, 5 mM desthiobiotin (Sigma-Aldrich); concentrated on WGA agarose beads (Vector Laboratories); and eluted with buffer containing 0.04% digitonin and 0.5 M N-acetyl-D-glucosamine. Protein purity was assessed using NuPAGE Bis-Tris gels (Invitrogen) with silver staining (Pierce). Monodispersity was determined by western blotting of NativePAGE Novex Bis-Tris Gels (Invitrogen) using anti-nicastrin (Sigma N1660) and anti-PS1-NTF (Abcam ab10281) antibodies and compared with NativeMark Unstained Protein Standard (Invitrogen LC0725).
Compound E-bound complexes were purified as above in 0.5–1.0 mM compound E during all steps.

Li Y., Lu S.H., Tsai C.J., Bohm C., Qamar S., Dodd R.B., Meadows W., Jeon A., McLeod A., Chen F., Arimon M., Berezovska O., Hyman B.T., Tomita T., Iwatsubo T., Johnson C.M., Farrer L.A., Schmitt-Ulms G., Fraser P.E, & St George-Hyslop P.H. (2014). Structural Interactions between Inhibitor and Substrate Docking Sites Give Insight into Mechanisms of Human PS1 Complexes. Structure(London, England:1993), 22(1), 125-135.

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Subcellular Localization of OIP5-AS1

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Cellular fractionation was performed to determine the subcellular localization of OIP5-AS1. Briefly, K1 and TPC-1 cells were lysed with RSB buffer (10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl₂, 100 mM NaCl) containing 4 mg/ml digitonin (BN2006, Thermo Fisher Scientific). After centrifugation, the supernatant was collected as the cytosolic extract. The remaining nuclear pellet was washed five times with RSB buffer and then lysed with RIPA buffer. GAPDH and U6 served as markers for the cytosolic and nuclear fraction, respectively.

Zhang X., Li D., Jia C., Cai H., Lv Z, & Wu B. (2021). METTL14 promotes tumorigenesis by regulating lncRNA OIP5-AS1/miR-98/ADAMTS8 signaling in papillary thyroid cancer. Cell Death & Disease, 12(6), 617.

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Proteinase-K Protection Assay for P2Y1 Receptor

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The proteinase-K protection assay was performed as described [25 (link), 26 (link)]. HeLa cells were plated in six-well culture dishes (4.0 X 10⁵ cells/well), grown overnight at 37°C and transfected with FLAG-P2Y₁ wildtype (WT) or FLAG-P2Y₁ Y155A, as described above. Cells were incubated with or without 10 μM ADP and 1 mM Leupeptin (Calbiochem). Cells were placed on ice and incubated for 5 min with PBS, harvested and gently permeabilized using 6.5 μg/ml digitonin (Thermo Fisher Scientific). Membranes were collected by centrifugation and resuspended in buffer (100 mM K₂HPO₄/KH₂PO₄, 5 mM MgCl₂, 250 mM sucrose). Membranes were then divided into three aliquots either left untreated, treated with 2.5 ng/ml proteinase-K, or treated with proteinase-K supplemented with 0.1% Triton-X 100 for 10 min at room temperature. After treatments, samples are diluted with 100 μl 2X SDS sample buffer containing 20 mM PMSF and analyzed by immunoblotting as described above.

Dores M.R., Grimsey N.J., Mendez F, & Trejo J. (2016). ALIX Regulates the Ubiquitin-Independent Lysosomal Sorting of the P2Y1 Purinergic Receptor via a YPX3L Motif. PLoS ONE, 11(6), e0157587.

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Co-Immunoprecipitation of Hydrogenosomal Proteins

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CoIPs were performed for the HA-tagged TvTom40-2 either with or without crosslinker using isolated hydrogenosomes from both WT and recombinant strains. For crosslinking, interacting proteins in hydrogenosomes (protein concentration 1 mg/mL) were crosslinked with 1 mM DSP (dithiobis(succinimidyl propionate); Thermo Scientific) for 30 minutes at 25 °C, excess DSP was quenched with 50 mM Tris (pH 7.5), and the hydrogenosomes were washed twice with isolation buffer. For coIP, the hydrogenosomes (protein concentration 1 mg/mL) were solubilised in MKG buffer (10 mM MOPS [3-(N-morpholino)propanesulfonic acid; pH 7], 50 mM potassium acetate, 10% glycerol, and EDTA-free cOmplete protease inhibitor cocktail [Roche]) containing 1% digitonin (Merck Millipore), and the clarified extract was incubated with Dynabeads (Thermo Fisher Scientific) coupled with α-HA antibody for 90 minutes on an overhead rotator at room temperature. The beads were washed thrice before elution with either SDS-PAGE buffer for crosslinking coIPs or elution buffer (MKG buffer with 0.25% digitonin and 1 mg/mL HA peptide, Thermo Fisher Scientific) for native coIPs. The coupling of α-HA antibody to the Dynabeads was performed according to the manufacturer’s instructions.

Makki A., Rada P., Žárský V., Kereïche S., Kováčik L., Novotný M., Jores T., Rapaport D, & Tachezy J. (2019). Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis. PLoS Biology, 17(1), e3000098.

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Digitonin-Mediated Permeabilization of HeLa Cells

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A single cell-derived clone of HeLa cells (ATCC, Manassas, VA; mycoplasma negative and validated by STR profiling) were maintained in OptiMEM (Gibco/ThermoFisher) with 4% FBS and plated on uncoated optical glass-bottom 96 well plates, at appropriate densities to reach 70–90% confluence on the day of the transport assay. To permeabilize, cells were rinsed for 2 min in ice-cold PBS, and permeabilized on ice for 10 min in 15–30 µg/mL digitonin (Calbiochem, San Diego, CA) in permeabilization buffer (PRB, 20 mM HEPES, 110 mM KOAc, 5 mM Mg(OAc)2, 0.5 mM EGTA, 250 mM sucrose, pH 7.5, with protease inhibitor cocktail). Following permeabilization, cells were placed on ice and rinsed 3 × 5 min in transport buffer (TRB, 20 mM HEPES, 110 mM KOAc, 2 mM Mg(OAc)₂, 5 mM NaOAc, 0.5 mM EGTA, 250 mM sucrose, pH 7.3, with protease inhibitor cocktail). The optimal digitonin concentration varied by cell density and passage number, and was optimized prior to each set of assays for the ability to permeabilize the majority of plasma membranes while maintaining nuclear exclusion of a 70 kD fluorescent dextran (ThermoFisher).

Hayes L.R., Duan L., Bowen K., Kalab P, & Rothstein J.D. (2020). C9orf72 arginine-rich dipeptide repeat proteins disrupt karyopherin-mediated nuclear import. eLife, 9, e51685.

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Native Mitochondrial Protein Complexes Analysis

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Isolated mitochondria (250 μg) of larvae tissues were suspended in BSA-free mitochondria isolation buffer (see above) and centrifuged at 10,000 × g at 4°C. Mitochondria (pellet) were resuspended in 100 μl 1X Native sample buffer (Invitrogen, BN20032) with either 1% Triton X-100 (Applichem, A4975) or 4% digitonin (Thermo Fischer Scientific, BN2006) and incubated for 10 min on ice before centrifuging at 20,000 × g at 4ºC for 30 min [80]. The supernatant (extracted solubilized complexes) was retained and Native PAGE sample additive G250 5% (Invitrogen, BN2004) was added. Samples were separated by 3–12% gradient BNGE and after electrophoresis the complexes were transferred on a polyvinylidene fluoride (PVDF) membrane and probed with the indicated antibodies.

Tsakiri E.N., Gumeni S., Vougas K., Pendin D., Papassideri I., Daga A., Gorgoulis V., Juhász G., Scorrano L, & Trougakos I.P. (2019). Proteasome dysfunction induces excessive proteome instability and loss of mitostasis that can be mitigated by enhancing mitochondrial fusion or autophagy. Autophagy, 15(10), 1757-1773.

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Cell Fractionation and Native PAGE Analysis

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HT29-SMART cells were untreated or treated with TNF, BV6, and zVAD in the absence or presence of NSA for the indicated times. Cell fractionation was performed as previously described^{52 (link)}. Briefly, cells were harvested with cold PBS and resuspended in a cell fractionation buffer (20 mM HEPES [pH 7.5], 100 mM KCl, 2.5 mM MgCl₂, and 100 mM sucrose) containing 0.025% digitonin (14592, Cayman Chemical), 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF, 1 μg ml⁻¹ aprotinin, 1 μg ml⁻¹ leupeptin, and 1 μg ml⁻¹ pepstatin on ice for 10 min. After centrifugation, the supernatant was collected as a cytosolic fraction. The resulting pellet was resuspended with the cell fractionation buffer described above and the final concentrations of digitonin were adjusted to 1% w/v and kept on ice for 20 min. After centrifugation, the 1% digitonin soluble membrane fraction and cytosolic fraction were subjected to 4–16% Bis–Tris Native PAGE gel (BN1002BOX, ThermoFisher) and then transferred onto PVDF membrane. In parallel experiments, membrane and cytosolic fractions were subjected to reducing SDS-PAGE and then transferred onto PVDF membrane. The membranes were analyzed as described in Western blotting.

Murai S., Yamaguchi Y., Shirasaki Y., Yamagishi M., Shindo R., Hildebrand J.M., Miura R., Nakabayashi O., Totsuka M., Tomida T., Adachi-Akahane S., Uemura S., Silke J., Yagita H., Miura M, & Nakano H. (2018). A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs. Nature Communications, 9, 4457.

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