Structured Review

Millipore digitonin
Digitonin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/digitonin/product/Millipore
Average 99 stars, based on 34 article reviews
Price from $9.99 to $1999.99
digitonin - by Bioz Stars, 2020-03
99/100 stars

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Centrifugation:

Article Title: CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10
Article Snippet: For immunoprecipitation experiments, cells were incubated with isolation buffer [20 m m HEPES (pH 7.4), 10% glycerol, HEPES and miniComplete protease inhibitor cocktail (Roche)] containing 1% w/v digitonin (Sigma) for 10 min on ice with periodic mixing by pipetting. .. Insoluble material was separated by centrifugation at 16 000 g at 4°C.

Article Title: Disordered clusters of Bak dimers rupture mitochondria during apoptosis
Article Snippet: Cells were washed in PBS and then re-suspended at a concentration of 1 × 107 cells/ml in wash buffer supplemented with digitonin (100 mM sucrose, 20 mM HEPES-NaOH pH 7.5, 100 mM KCl, 2.5 mM MgCl2 , 4 μg/ml Pepstatin A (Sigma-Aldrich, St. Louis, MO, USA), Complete protease inhibitors (Roche, Castle Hill, NSW, Australia) and 0.025% w/v digitonin (Calbiochem, Merck, Darmstadt, Germany). .. Cells were incubated for 10 min on ice, then membrane fractions were collected by centrifugation at 16,000 ×g for 5 min and the supernatant discarded.

Article Title: Cyclophilin D deficiency attenuates mitochondrial F1Fo ATP synthase dysfunction via OSCP in Alzheimer’s disease
Article Snippet: .. The pellet was collected and resuspended in isolation buffer with 0.02% Digitonin (Sigma-Aldrich) and then subjected to the second centrifugation at 8,000 x g for another 10 minutes. .. The pellet was then washed by additional centrifugation step in ice-cold isolation buffer without EGTA for experiments.

Luciferase:

Article Title: Human Cytomegalovirus DNA Polymerase Subunit UL44 Antagonizes Antiviral Immune Responses by Suppressing IRF3- and NF-κB-Mediated Transcription
Article Snippet: .. Lipofectamine 2000 (Invitrogen), Polybrene (Millipore), puromycin and RNase inhibitor (Thermo), the Dual-Specific Luciferase Assay kit (Promega), SYBR green (Bio-Rad), digitonin (Sigma), and mouse antibodies against Flag and β-actin (Sigma), hemagglutinin (HA) (Covance), phospho-NF-κB p65, phospho-Stat1 (Tyr 701) (9167), and phospho-IRF-3 (4947S) (Cell Signaling Technology), phospho-TBK1 (ab109272) and TBK1 (ab40676) (Abcam), IRF3 (sc-9082), and STAT1 (sc-346) (Santa Cruz Biotechnology) were purchased from their respective manufacturers. .. Antisera against UL44 and UL82 were generated by immunizing rabbits or mice with purified recombinant UL44 or UL82.

Bradford Assay:

Article Title: Cyclophilin D deficiency attenuates mitochondrial F1Fo ATP synthase dysfunction via OSCP in Alzheimer’s disease
Article Snippet: The pellet was collected and resuspended in isolation buffer with 0.02% Digitonin (Sigma-Aldrich) and then subjected to the second centrifugation at 8,000 x g for another 10 minutes. .. Protein concentrations were measured using Bradford assay for protein detection (BioRad).

SYBR Green Assay:

Article Title: Human Cytomegalovirus DNA Polymerase Subunit UL44 Antagonizes Antiviral Immune Responses by Suppressing IRF3- and NF-κB-Mediated Transcription
Article Snippet: .. Lipofectamine 2000 (Invitrogen), Polybrene (Millipore), puromycin and RNase inhibitor (Thermo), the Dual-Specific Luciferase Assay kit (Promega), SYBR green (Bio-Rad), digitonin (Sigma), and mouse antibodies against Flag and β-actin (Sigma), hemagglutinin (HA) (Covance), phospho-NF-κB p65, phospho-Stat1 (Tyr 701) (9167), and phospho-IRF-3 (4947S) (Cell Signaling Technology), phospho-TBK1 (ab109272) and TBK1 (ab40676) (Abcam), IRF3 (sc-9082), and STAT1 (sc-346) (Santa Cruz Biotechnology) were purchased from their respective manufacturers. .. Antisera against UL44 and UL82 were generated by immunizing rabbits or mice with purified recombinant UL44 or UL82.

Incubation:

Article Title: The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6.
Article Snippet: After washing with PBS containing 0.1% Tween 20, cells were incubated with Alexa Fluor 568, 488-conjugated secondary antibodies (Molecular Probes) for 1 h at room temperature. .. For total permeabilization, cells were permeabilized with 0.05% saponin (Sigma) for 10 min at 20°C, whereas selective permeabilization was performed for 15 min at 4°C with 0.000125% digitonin (Calbiochem) in a buffer containing 10 mM HEPES (pH 6.8), 1 mM EDTA, 0.3 M sucrose, 0.1 M KCl, and 2.5 mM MgCl 2 .

Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
Article Snippet: .. U2OS cells were plated on sterile coverslips, cultured under the conditions indicated and fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS; Gibco, Life Technologies, 14190–094) buffer, for 20 min. For immunofluorescent staining of endogenous ULK1, SQSTM1 and MAP1LC3B, after fixation, the cells were permeabilized with digitonin (50 µg/µl; Sigma-Aldrich, 11024-24-1) in PBS for 10 min and blocked by incubation with 5% bovine serum albumin (Sigma-Aldrich, 9048-46-8) in PBS for 1 h at room temperature, followed by incubation with the primary antibodies for 2 h at 37°C. .. After washing 3 times with PBS, coverslips were embedded in Mowiol 4–88 (Carl Roth GmbH + Co. KG, 0713.1) containing 1 mg/ml Hoechst (Life Technologies, H1399).

Article Title: CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10
Article Snippet: .. For immunoprecipitation experiments, cells were incubated with isolation buffer [20 m m HEPES (pH 7.4), 10% glycerol, HEPES and miniComplete protease inhibitor cocktail (Roche)] containing 1% w/v digitonin (Sigma) for 10 min on ice with periodic mixing by pipetting. .. Insoluble material was separated by centrifugation at 16 000 g at 4°C.

Article Title: Disordered clusters of Bak dimers rupture mitochondria during apoptosis
Article Snippet: Cells were washed in PBS and then re-suspended at a concentration of 1 × 107 cells/ml in wash buffer supplemented with digitonin (100 mM sucrose, 20 mM HEPES-NaOH pH 7.5, 100 mM KCl, 2.5 mM MgCl2 , 4 μg/ml Pepstatin A (Sigma-Aldrich, St. Louis, MO, USA), Complete protease inhibitors (Roche, Castle Hill, NSW, Australia) and 0.025% w/v digitonin (Calbiochem, Merck, Darmstadt, Germany). .. Cells were incubated for 10 min on ice, then membrane fractions were collected by centrifugation at 16,000 ×g for 5 min and the supernatant discarded.

Article Title: STRUCTURAL BASIS OF Bcl-xL RECOGNITION BY A BH3-MIMETIC ?/?-PEPTIDE GENERATED VIA SEQUENCE-BASED DESIGN
Article Snippet: Mouse embryonic fibroblasts (wild-type, mcl-1 -/- and bcl-x -/- ) (~2 × 106 cells) were permeabilized in digitonin-containing permeabilization solution (0.05% (w/v) digitonin (Calbiochem) in 20 mM HEPES pH 7.2, 100 mM KCl, 5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 250 mM sucrose), supplemented with protease inhibitors (Roche), for 10 minutes on ice. .. The mitochondria-containing crude lysates were incubated with peptide (10 μM) at 30° C for 1 hour before pelleting.

Expressing:

Article Title: Disordered clusters of Bak dimers rupture mitochondria during apoptosis
Article Snippet: Preparation of membrane fractions enriched for mitochondria Bak−/− Bax−/− MEFs expressing Bak cysteine mutants at levels comparable to endogenous mouse Bak ( ) were permeabilised and membrane fractions containing the mitochondria were isolated as previously described ( ). .. Cells were washed in PBS and then re-suspended at a concentration of 1 × 107 cells/ml in wash buffer supplemented with digitonin (100 mM sucrose, 20 mM HEPES-NaOH pH 7.5, 100 mM KCl, 2.5 mM MgCl2 , 4 μg/ml Pepstatin A (Sigma-Aldrich, St. Louis, MO, USA), Complete protease inhibitors (Roche, Castle Hill, NSW, Australia) and 0.025% w/v digitonin (Calbiochem, Merck, Darmstadt, Germany).

Release Assay:

Article Title: STRUCTURAL BASIS OF Bcl-xL RECOGNITION BY A BH3-MIMETIC ?/?-PEPTIDE GENERATED VIA SEQUENCE-BASED DESIGN
Article Snippet: Paragraph title: Cytochrome c release assay ... Mouse embryonic fibroblasts (wild-type, mcl-1 -/- and bcl-x -/- ) (~2 × 106 cells) were permeabilized in digitonin-containing permeabilization solution (0.05% (w/v) digitonin (Calbiochem) in 20 mM HEPES pH 7.2, 100 mM KCl, 5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 250 mM sucrose), supplemented with protease inhibitors (Roche), for 10 minutes on ice.

Transfection:

Article Title: The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6.
Article Snippet: Confocal immunofluorescence microscopy Vero E6 or HeLa cells transfected in multiwell chambers were fixed with 4% paraformaldehyde in PBS and then permeabilized with cold methanol for 5 min at room temperature. .. For total permeabilization, cells were permeabilized with 0.05% saponin (Sigma) for 10 min at 20°C, whereas selective permeabilization was performed for 15 min at 4°C with 0.000125% digitonin (Calbiochem) in a buffer containing 10 mM HEPES (pH 6.8), 1 mM EDTA, 0.3 M sucrose, 0.1 M KCl, and 2.5 mM MgCl 2 .

Concentration Assay:

Article Title: CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10
Article Snippet: Tris (pH 8.0) was added for a final concentration of 5 m m to quench remaining crosslinker and the cells were incubated for a further 15 min at room temperature. .. For immunoprecipitation experiments, cells were incubated with isolation buffer [20 m m HEPES (pH 7.4), 10% glycerol, HEPES and miniComplete protease inhibitor cocktail (Roche)] containing 1% w/v digitonin (Sigma) for 10 min on ice with periodic mixing by pipetting.

Article Title: Disordered clusters of Bak dimers rupture mitochondria during apoptosis
Article Snippet: .. Cells were washed in PBS and then re-suspended at a concentration of 1 × 107 cells/ml in wash buffer supplemented with digitonin (100 mM sucrose, 20 mM HEPES-NaOH pH 7.5, 100 mM KCl, 2.5 mM MgCl2 , 4 μg/ml Pepstatin A (Sigma-Aldrich, St. Louis, MO, USA), Complete protease inhibitors (Roche, Castle Hill, NSW, Australia) and 0.025% w/v digitonin (Calbiochem, Merck, Darmstadt, Germany). .. Cells were incubated for 10 min on ice, then membrane fractions were collected by centrifugation at 16,000 ×g for 5 min and the supernatant discarded.

Protease Inhibitor:

Article Title: CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10
Article Snippet: .. For immunoprecipitation experiments, cells were incubated with isolation buffer [20 m m HEPES (pH 7.4), 10% glycerol, HEPES and miniComplete protease inhibitor cocktail (Roche)] containing 1% w/v digitonin (Sigma) for 10 min on ice with periodic mixing by pipetting. .. Insoluble material was separated by centrifugation at 16 000 g at 4°C.

Cell Culture:

Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
Article Snippet: .. U2OS cells were plated on sterile coverslips, cultured under the conditions indicated and fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS; Gibco, Life Technologies, 14190–094) buffer, for 20 min. For immunofluorescent staining of endogenous ULK1, SQSTM1 and MAP1LC3B, after fixation, the cells were permeabilized with digitonin (50 µg/µl; Sigma-Aldrich, 11024-24-1) in PBS for 10 min and blocked by incubation with 5% bovine serum albumin (Sigma-Aldrich, 9048-46-8) in PBS for 1 h at room temperature, followed by incubation with the primary antibodies for 2 h at 37°C. .. After washing 3 times with PBS, coverslips were embedded in Mowiol 4–88 (Carl Roth GmbH + Co. KG, 0713.1) containing 1 mg/ml Hoechst (Life Technologies, H1399).

Generated:

Article Title: Human Cytomegalovirus DNA Polymerase Subunit UL44 Antagonizes Antiviral Immune Responses by Suppressing IRF3- and NF-κB-Mediated Transcription
Article Snippet: Lipofectamine 2000 (Invitrogen), Polybrene (Millipore), puromycin and RNase inhibitor (Thermo), the Dual-Specific Luciferase Assay kit (Promega), SYBR green (Bio-Rad), digitonin (Sigma), and mouse antibodies against Flag and β-actin (Sigma), hemagglutinin (HA) (Covance), phospho-NF-κB p65, phospho-Stat1 (Tyr 701) (9167), and phospho-IRF-3 (4947S) (Cell Signaling Technology), phospho-TBK1 (ab109272) and TBK1 (ab40676) (Abcam), IRF3 (sc-9082), and STAT1 (sc-346) (Santa Cruz Biotechnology) were purchased from their respective manufacturers. .. Antisera against UL44 and UL82 were generated by immunizing rabbits or mice with purified recombinant UL44 or UL82.

other:

Article Title: Pathogenic Variants in NUP214 Cause “Plugged” Nuclear Pore Channels and Acute Febrile Encephalopathy
Article Snippet: The coverslips were washed once in cold PBS, once in cold transport buffer (TB; 20 mM HEPES [pH 7.3], 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EDTA, 2 mM DTT, 1 mM PMFS, and 1 μg/mL each of aprotinin and leupeptin) and the cells were permeabilized in 50 μL TB supplemented with 0.005% digitonin (CAS 11024-24-1, Calbiochem) for 7 min on ice.

Article Title: Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP–AMP
Article Snippet: After an additional 14-h incubation in medium, luciferase activity was determined following the standard protocol (Promega, E1501).

Imaging:

Article Title: CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10
Article Snippet: Blots were imaged for chemiluminescent using the ChemiDoc Imaging system (Biorad) or the Odyssey Imaging System (LiCor). .. For immunoprecipitation experiments, cells were incubated with isolation buffer [20 m m HEPES (pH 7.4), 10% glycerol, HEPES and miniComplete protease inhibitor cocktail (Roche)] containing 1% w/v digitonin (Sigma) for 10 min on ice with periodic mixing by pipetting.

Recombinant:

Article Title: Effect of N-(2-Aminoethyl) Ethanolamine on Hypertrophic Scarring Changes in Vitro: Finding Novel Anti-Fibrotic Therapies
Article Snippet: We purchased the following from Sigma-Aldrich (St. Louis, MO): the AEEA analogs diethylenetriamine (catalog # D93856), diethanolamine (catalog # D8885), 2-(2-(methylamino)-ethylamino)-ethanol (catalog # S522449), triethanolamine (catalog # 90279), 2-(2-aminoethoxy)ethanol (catalog # ), 2-(2-aminoethoxy)ethylamine (catalog # CDS000024); digitonin (catalog # 260746 Aldrich), sodium deoxycholate (catalog # D6750); (+)-sodium L-ascorbate (catalog # A4034); sodium phosphate dibasic (catalog # S0876); sodium phosphate mono basic (catalog # S0751); Trizma base (catalog # T-8524); Tween 20 (catalog # P9416); and urea (catalog # U5128), and chloramine T (catalog # 857319). .. Recombinant human TGF-β1 protein (catalog# GF111) was purchased from EMD Millipore.

Article Title: Human Cytomegalovirus DNA Polymerase Subunit UL44 Antagonizes Antiviral Immune Responses by Suppressing IRF3- and NF-κB-Mediated Transcription
Article Snippet: Lipofectamine 2000 (Invitrogen), Polybrene (Millipore), puromycin and RNase inhibitor (Thermo), the Dual-Specific Luciferase Assay kit (Promega), SYBR green (Bio-Rad), digitonin (Sigma), and mouse antibodies against Flag and β-actin (Sigma), hemagglutinin (HA) (Covance), phospho-NF-κB p65, phospho-Stat1 (Tyr 701) (9167), and phospho-IRF-3 (4947S) (Cell Signaling Technology), phospho-TBK1 (ab109272) and TBK1 (ab40676) (Abcam), IRF3 (sc-9082), and STAT1 (sc-346) (Santa Cruz Biotechnology) were purchased from their respective manufacturers. .. Antisera against UL44 and UL82 were generated by immunizing rabbits or mice with purified recombinant UL44 or UL82.

Immunofluorescence:

Article Title: The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6.
Article Snippet: Paragraph title: Confocal immunofluorescence microscopy ... For total permeabilization, cells were permeabilized with 0.05% saponin (Sigma) for 10 min at 20°C, whereas selective permeabilization was performed for 15 min at 4°C with 0.000125% digitonin (Calbiochem) in a buffer containing 10 mM HEPES (pH 6.8), 1 mM EDTA, 0.3 M sucrose, 0.1 M KCl, and 2.5 mM MgCl 2 .

Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
Article Snippet: U2OS cells were plated on sterile coverslips, cultured under the conditions indicated and fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS; Gibco, Life Technologies, 14190–094) buffer, for 20 min. For immunofluorescent staining of endogenous ULK1, SQSTM1 and MAP1LC3B, after fixation, the cells were permeabilized with digitonin (50 µg/µl; Sigma-Aldrich, 11024-24-1) in PBS for 10 min and blocked by incubation with 5% bovine serum albumin (Sigma-Aldrich, 9048-46-8) in PBS for 1 h at room temperature, followed by incubation with the primary antibodies for 2 h at 37°C. .. After rehydration in PBS, a standard protocol for immunofluorescence was followed [ ].

Magnetic Beads:

Article Title: CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10
Article Snippet: For immunoprecipitation experiments, cells were incubated with isolation buffer [20 m m HEPES (pH 7.4), 10% glycerol, HEPES and miniComplete protease inhibitor cocktail (Roche)] containing 1% w/v digitonin (Sigma) for 10 min on ice with periodic mixing by pipetting. .. A total of 10% was retained as input and the remainder was incubated with 40 μL anti-Flag M2 magnetic beads (Sigma) with end-over-end mixing at 4°C for 2–3 h. Beads were washed three times with isolation buffer containing 0.05% digitonin.

Isolation:

Article Title: CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10
Article Snippet: .. For immunoprecipitation experiments, cells were incubated with isolation buffer [20 m m HEPES (pH 7.4), 10% glycerol, HEPES and miniComplete protease inhibitor cocktail (Roche)] containing 1% w/v digitonin (Sigma) for 10 min on ice with periodic mixing by pipetting. .. Insoluble material was separated by centrifugation at 16 000 g at 4°C.

Article Title: Disordered clusters of Bak dimers rupture mitochondria during apoptosis
Article Snippet: Preparation of membrane fractions enriched for mitochondria Bak−/− Bax−/− MEFs expressing Bak cysteine mutants at levels comparable to endogenous mouse Bak ( ) were permeabilised and membrane fractions containing the mitochondria were isolated as previously described ( ). .. Cells were washed in PBS and then re-suspended at a concentration of 1 × 107 cells/ml in wash buffer supplemented with digitonin (100 mM sucrose, 20 mM HEPES-NaOH pH 7.5, 100 mM KCl, 2.5 mM MgCl2 , 4 μg/ml Pepstatin A (Sigma-Aldrich, St. Louis, MO, USA), Complete protease inhibitors (Roche, Castle Hill, NSW, Australia) and 0.025% w/v digitonin (Calbiochem, Merck, Darmstadt, Germany).

Article Title: Cyclophilin D deficiency attenuates mitochondrial F1Fo ATP synthase dysfunction via OSCP in Alzheimer’s disease
Article Snippet: .. The pellet was collected and resuspended in isolation buffer with 0.02% Digitonin (Sigma-Aldrich) and then subjected to the second centrifugation at 8,000 x g for another 10 minutes. .. The pellet was then washed by additional centrifugation step in ice-cold isolation buffer without EGTA for experiments.

Microscopy:

Article Title: The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6.
Article Snippet: Paragraph title: Confocal immunofluorescence microscopy ... For total permeabilization, cells were permeabilized with 0.05% saponin (Sigma) for 10 min at 20°C, whereas selective permeabilization was performed for 15 min at 4°C with 0.000125% digitonin (Calbiochem) in a buffer containing 10 mM HEPES (pH 6.8), 1 mM EDTA, 0.3 M sucrose, 0.1 M KCl, and 2.5 mM MgCl 2 .

Mouse Assay:

Article Title: Human Cytomegalovirus DNA Polymerase Subunit UL44 Antagonizes Antiviral Immune Responses by Suppressing IRF3- and NF-κB-Mediated Transcription
Article Snippet: Lipofectamine 2000 (Invitrogen), Polybrene (Millipore), puromycin and RNase inhibitor (Thermo), the Dual-Specific Luciferase Assay kit (Promega), SYBR green (Bio-Rad), digitonin (Sigma), and mouse antibodies against Flag and β-actin (Sigma), hemagglutinin (HA) (Covance), phospho-NF-κB p65, phospho-Stat1 (Tyr 701) (9167), and phospho-IRF-3 (4947S) (Cell Signaling Technology), phospho-TBK1 (ab109272) and TBK1 (ab40676) (Abcam), IRF3 (sc-9082), and STAT1 (sc-346) (Santa Cruz Biotechnology) were purchased from their respective manufacturers. .. Antisera against UL44 and UL82 were generated by immunizing rabbits or mice with purified recombinant UL44 or UL82.

Confocal Microscopy:

Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
Article Snippet: Paragraph title: Confocal microscopy analysis ... U2OS cells were plated on sterile coverslips, cultured under the conditions indicated and fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS; Gibco, Life Technologies, 14190–094) buffer, for 20 min. For immunofluorescent staining of endogenous ULK1, SQSTM1 and MAP1LC3B, after fixation, the cells were permeabilized with digitonin (50 µg/µl; Sigma-Aldrich, 11024-24-1) in PBS for 10 min and blocked by incubation with 5% bovine serum albumin (Sigma-Aldrich, 9048-46-8) in PBS for 1 h at room temperature, followed by incubation with the primary antibodies for 2 h at 37°C.

Staining:

Article Title: USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy
Article Snippet: .. U2OS cells were plated on sterile coverslips, cultured under the conditions indicated and fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS; Gibco, Life Technologies, 14190–094) buffer, for 20 min. For immunofluorescent staining of endogenous ULK1, SQSTM1 and MAP1LC3B, after fixation, the cells were permeabilized with digitonin (50 µg/µl; Sigma-Aldrich, 11024-24-1) in PBS for 10 min and blocked by incubation with 5% bovine serum albumin (Sigma-Aldrich, 9048-46-8) in PBS for 1 h at room temperature, followed by incubation with the primary antibodies for 2 h at 37°C. .. After washing 3 times with PBS, coverslips were embedded in Mowiol 4–88 (Carl Roth GmbH + Co. KG, 0713.1) containing 1 mg/ml Hoechst (Life Technologies, H1399).

Purification:

Article Title: Human Cytomegalovirus DNA Polymerase Subunit UL44 Antagonizes Antiviral Immune Responses by Suppressing IRF3- and NF-κB-Mediated Transcription
Article Snippet: Lipofectamine 2000 (Invitrogen), Polybrene (Millipore), puromycin and RNase inhibitor (Thermo), the Dual-Specific Luciferase Assay kit (Promega), SYBR green (Bio-Rad), digitonin (Sigma), and mouse antibodies against Flag and β-actin (Sigma), hemagglutinin (HA) (Covance), phospho-NF-κB p65, phospho-Stat1 (Tyr 701) (9167), and phospho-IRF-3 (4947S) (Cell Signaling Technology), phospho-TBK1 (ab109272) and TBK1 (ab40676) (Abcam), IRF3 (sc-9082), and STAT1 (sc-346) (Santa Cruz Biotechnology) were purchased from their respective manufacturers. .. Antisera against UL44 and UL82 were generated by immunizing rabbits or mice with purified recombinant UL44 or UL82.

SDS Page:

Article Title: CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10
Article Snippet: Cells were subsequently lysed and subjected to SDS-PAGE separation and immunoblotting as described above. .. For immunoprecipitation experiments, cells were incubated with isolation buffer [20 m m HEPES (pH 7.4), 10% glycerol, HEPES and miniComplete protease inhibitor cocktail (Roche)] containing 1% w/v digitonin (Sigma) for 10 min on ice with periodic mixing by pipetting.

Article Title: STRUCTURAL BASIS OF Bcl-xL RECOGNITION BY A BH3-MIMETIC ?/?-PEPTIDE GENERATED VIA SEQUENCE-BASED DESIGN
Article Snippet: Mouse embryonic fibroblasts (wild-type, mcl-1 -/- and bcl-x -/- ) (~2 × 106 cells) were permeabilized in digitonin-containing permeabilization solution (0.05% (w/v) digitonin (Calbiochem) in 20 mM HEPES pH 7.2, 100 mM KCl, 5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 250 mM sucrose), supplemented with protease inhibitors (Roche), for 10 minutes on ice. .. Proteins were resolved by SDS-PAGE and transferred onto nitrocellulose membranes.

Confocal Laser Scanning Microscopy:

Article Title: The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6.
Article Snippet: The slides were then mounted with Prolong Gold anti-fade reagent (Molecular Probes) and visualized by confocal laser scanning microscopy (Zeiss, LSM 510). .. For total permeabilization, cells were permeabilized with 0.05% saponin (Sigma) for 10 min at 20°C, whereas selective permeabilization was performed for 15 min at 4°C with 0.000125% digitonin (Calbiochem) in a buffer containing 10 mM HEPES (pH 6.8), 1 mM EDTA, 0.3 M sucrose, 0.1 M KCl, and 2.5 mM MgCl 2 .

Immunoprecipitation:

Article Title: CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10
Article Snippet: .. For immunoprecipitation experiments, cells were incubated with isolation buffer [20 m m HEPES (pH 7.4), 10% glycerol, HEPES and miniComplete protease inhibitor cocktail (Roche)] containing 1% w/v digitonin (Sigma) for 10 min on ice with periodic mixing by pipetting. .. Insoluble material was separated by centrifugation at 16 000 g at 4°C.

Fractionation:

Article Title: CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10
Article Snippet: Paragraph title: Immunoblot analysis, subcellular fractionation, crosslinking experiments and immunoprecipitation experiments ... For immunoprecipitation experiments, cells were incubated with isolation buffer [20 m m HEPES (pH 7.4), 10% glycerol, HEPES and miniComplete protease inhibitor cocktail (Roche)] containing 1% w/v digitonin (Sigma) for 10 min on ice with periodic mixing by pipetting.

Lysis:

Article Title: STRUCTURAL BASIS OF Bcl-xL RECOGNITION BY A BH3-MIMETIC ?/?-PEPTIDE GENERATED VIA SEQUENCE-BASED DESIGN
Article Snippet: Mouse embryonic fibroblasts (wild-type, mcl-1 -/- and bcl-x -/- ) (~2 × 106 cells) were permeabilized in digitonin-containing permeabilization solution (0.05% (w/v) digitonin (Calbiochem) in 20 mM HEPES pH 7.2, 100 mM KCl, 5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 250 mM sucrose), supplemented with protease inhibitors (Roche), for 10 minutes on ice. .. The supernatant was retained as the soluble fraction while the pellet, which contained intact mitochondria, was solubilized in lysis buffer (1% (v/v) Triton-X-100 in 20 mM Tris-pH 7.4, 135 mM NaCl, 1.5 mM MgCl2 , 1 mM EDTA, 10% (v/v) glycerol) supplemented with protease inhibitors (Roche).

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  • 77
    Millipore digitonin proteinase k assay
    Li TXN3 is an integral membrane protein with the N-terminal domain exposed to the cytosol. A. Alkaline carbonate extraction performed on 6His Li TXN3-expressing L. infantum promastigotes. Parasites were disrupted in either 10 mM Tris-HCl pH 7.4, 10 mM Tris-HCl 0.5 M NaCl pH 7.4, or 0.1 M Na 2 CO 3 pH 11.5 and fractionated by ultracentrifugation. Total protein extracts (T), as well as supernatants (S) and membrane pellets (P) resulting from fractionation were analyzed by western blot using anti-His antibody to detect 6His Li TXN3. Anti-gp63 and anti- Li cTXNPx2 antibodies were used to control for membrane and hydrophilic proteins, respectively. B. Protein samples resulting from permeabilization of 6His Li TXN3-expressing promastigotes with increasing concentrations of digitonin in the presence of <t>proteinase</t> K were analyzed by western blot using an anti-His antibody. Other antibodies were used to detect the cytosolic protein Li cTXNPx2, as well as a mitochondrial intermembrane space molecule (cytochrome c, detected with anti- Tb Cytc).
    Digitonin Proteinase K Assay, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/digitonin proteinase k assay/product/Millipore
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    digitonin proteinase k assay - by Bioz Stars, 2020-03
    77/100 stars
      Buy from Supplier

    87
    Millipore high purity digitonin
    Li TXN3 is an integral membrane protein with the N-terminal domain exposed to the cytosol. A. Alkaline carbonate extraction performed on 6His Li TXN3-expressing L. infantum promastigotes. Parasites were disrupted in either 10 mM Tris-HCl pH 7.4, 10 mM Tris-HCl 0.5 M NaCl pH 7.4, or 0.1 M Na 2 CO 3 pH 11.5 and fractionated by ultracentrifugation. Total protein extracts (T), as well as supernatants (S) and membrane pellets (P) resulting from fractionation were analyzed by western blot using anti-His antibody to detect 6His Li TXN3. Anti-gp63 and anti- Li cTXNPx2 antibodies were used to control for membrane and hydrophilic proteins, respectively. B. Protein samples resulting from permeabilization of 6His Li TXN3-expressing promastigotes with increasing concentrations of digitonin in the presence of <t>proteinase</t> K were analyzed by western blot using an anti-His antibody. Other antibodies were used to detect the cytosolic protein Li cTXNPx2, as well as a mitochondrial intermembrane space molecule (cytochrome c, detected with anti- Tb Cytc).
    High Purity Digitonin, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high purity digitonin/product/Millipore
    Average 87 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    high purity digitonin - by Bioz Stars, 2020-03
    87/100 stars
      Buy from Supplier

    87
    Millipore digitonin stock
    Li TXN3 is an integral membrane protein with the N-terminal domain exposed to the cytosol. A. Alkaline carbonate extraction performed on 6His Li TXN3-expressing L. infantum promastigotes. Parasites were disrupted in either 10 mM Tris-HCl pH 7.4, 10 mM Tris-HCl 0.5 M NaCl pH 7.4, or 0.1 M Na 2 CO 3 pH 11.5 and fractionated by ultracentrifugation. Total protein extracts (T), as well as supernatants (S) and membrane pellets (P) resulting from fractionation were analyzed by western blot using anti-His antibody to detect 6His Li TXN3. Anti-gp63 and anti- Li cTXNPx2 antibodies were used to control for membrane and hydrophilic proteins, respectively. B. Protein samples resulting from permeabilization of 6His Li TXN3-expressing promastigotes with increasing concentrations of digitonin in the presence of <t>proteinase</t> K were analyzed by western blot using an anti-His antibody. Other antibodies were used to detect the cytosolic protein Li cTXNPx2, as well as a mitochondrial intermembrane space molecule (cytochrome c, detected with anti- Tb Cytc).
    Digitonin Stock, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    digitonin stock - by Bioz Stars, 2020-03
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    80
    Millipore phenol red digitonin
    Li TXN3 is an integral membrane protein with the N-terminal domain exposed to the cytosol. A. Alkaline carbonate extraction performed on 6His Li TXN3-expressing L. infantum promastigotes. Parasites were disrupted in either 10 mM Tris-HCl pH 7.4, 10 mM Tris-HCl 0.5 M NaCl pH 7.4, or 0.1 M Na 2 CO 3 pH 11.5 and fractionated by ultracentrifugation. Total protein extracts (T), as well as supernatants (S) and membrane pellets (P) resulting from fractionation were analyzed by western blot using anti-His antibody to detect 6His Li TXN3. Anti-gp63 and anti- Li cTXNPx2 antibodies were used to control for membrane and hydrophilic proteins, respectively. B. Protein samples resulting from permeabilization of 6His Li TXN3-expressing promastigotes with increasing concentrations of digitonin in the presence of <t>proteinase</t> K were analyzed by western blot using an anti-His antibody. Other antibodies were used to detect the cytosolic protein Li cTXNPx2, as well as a mitochondrial intermembrane space molecule (cytochrome c, detected with anti- Tb Cytc).
    Phenol Red Digitonin, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Li TXN3 is an integral membrane protein with the N-terminal domain exposed to the cytosol. A. Alkaline carbonate extraction performed on 6His Li TXN3-expressing L. infantum promastigotes. Parasites were disrupted in either 10 mM Tris-HCl pH 7.4, 10 mM Tris-HCl 0.5 M NaCl pH 7.4, or 0.1 M Na 2 CO 3 pH 11.5 and fractionated by ultracentrifugation. Total protein extracts (T), as well as supernatants (S) and membrane pellets (P) resulting from fractionation were analyzed by western blot using anti-His antibody to detect 6His Li TXN3. Anti-gp63 and anti- Li cTXNPx2 antibodies were used to control for membrane and hydrophilic proteins, respectively. B. Protein samples resulting from permeabilization of 6His Li TXN3-expressing promastigotes with increasing concentrations of digitonin in the presence of proteinase K were analyzed by western blot using an anti-His antibody. Other antibodies were used to detect the cytosolic protein Li cTXNPx2, as well as a mitochondrial intermembrane space molecule (cytochrome c, detected with anti- Tb Cytc).

    Journal: PLoS ONE

    Article Title: Mitochondrial Redox Metabolism in Trypanosomatids Is Independent of Tryparedoxin Activity

    doi: 10.1371/journal.pone.0012607

    Figure Lengend Snippet: Li TXN3 is an integral membrane protein with the N-terminal domain exposed to the cytosol. A. Alkaline carbonate extraction performed on 6His Li TXN3-expressing L. infantum promastigotes. Parasites were disrupted in either 10 mM Tris-HCl pH 7.4, 10 mM Tris-HCl 0.5 M NaCl pH 7.4, or 0.1 M Na 2 CO 3 pH 11.5 and fractionated by ultracentrifugation. Total protein extracts (T), as well as supernatants (S) and membrane pellets (P) resulting from fractionation were analyzed by western blot using anti-His antibody to detect 6His Li TXN3. Anti-gp63 and anti- Li cTXNPx2 antibodies were used to control for membrane and hydrophilic proteins, respectively. B. Protein samples resulting from permeabilization of 6His Li TXN3-expressing promastigotes with increasing concentrations of digitonin in the presence of proteinase K were analyzed by western blot using an anti-His antibody. Other antibodies were used to detect the cytosolic protein Li cTXNPx2, as well as a mitochondrial intermembrane space molecule (cytochrome c, detected with anti- Tb Cytc).

    Article Snippet: Digitonin/proteinase K assay Twelve and a half million promastigotes (about 25 µg of total protein) suspended in 18.75 µl DIG buffer (25 mM Tris, pH 7.5, 1 mM EDTA, 0.6 M sucrose) containing 50 µg ml−1 proteinase K (PK), were permeabilized by addition of 6.25 µl of prediluted digitonin (Calbiochem) to final concentrations of 0–2 mg of digitonin per mg of cellular protein.

    Techniques: Expressing, Fractionation, Western Blot

    The cytosolic Li TXN1 enzyme is not present in the mitochondrion of txn2 − promastigotes. Protein samples resulting from the permeabilization of txn2 ù promastigotes with increasing concentrations of digitonin in the presence of proteinase K were analyzed by western blot using anti- Li TXN1 sera. Anti- Li cTXNPx2 and anti- Li mTXNPx were employed to detect cytosolic and mitochondrial compartments, respectively.

    Journal: PLoS ONE

    Article Title: Mitochondrial Redox Metabolism in Trypanosomatids Is Independent of Tryparedoxin Activity

    doi: 10.1371/journal.pone.0012607

    Figure Lengend Snippet: The cytosolic Li TXN1 enzyme is not present in the mitochondrion of txn2 − promastigotes. Protein samples resulting from the permeabilization of txn2 ù promastigotes with increasing concentrations of digitonin in the presence of proteinase K were analyzed by western blot using anti- Li TXN1 sera. Anti- Li cTXNPx2 and anti- Li mTXNPx were employed to detect cytosolic and mitochondrial compartments, respectively.

    Article Snippet: Digitonin/proteinase K assay Twelve and a half million promastigotes (about 25 µg of total protein) suspended in 18.75 µl DIG buffer (25 mM Tris, pH 7.5, 1 mM EDTA, 0.6 M sucrose) containing 50 µg ml−1 proteinase K (PK), were permeabilized by addition of 6.25 µl of prediluted digitonin (Calbiochem) to final concentrations of 0–2 mg of digitonin per mg of cellular protein.

    Techniques: Western Blot