protk  (New England Biolabs)


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    Name:
    Proteinase K Molecular Biology Grade
    Description:
    Proteinase K Molecular Biology Grade 2 ml
    Catalog Number:
    P8107S
    Price:
    83
    Category:
    Proteases
    Size:
    2 ml
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    Structured Review

    New England Biolabs protk
    Proteinase K Molecular Biology Grade
    Proteinase K Molecular Biology Grade 2 ml
    https://www.bioz.com/result/protk/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protk - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "High-Bandwidth Protein Analysis Using Solid-State Nanopores"

    Article Title: High-Bandwidth Protein Analysis Using Solid-State Nanopores

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2013.12.025

    ( a and b ) Dwell-time distributions for ProtK ( a ) and RNase ( b ) at selected voltages, along with fits to Eq. 1 with D and v as free parameters ( black line ) and constrained fits for bulk D 0 values ( dashed red line ). Missed regimes shaded red in distributions. ( c ) Diffusion coefficients ( D ) obtained for the proteins from the fits to Eq. 1. ( d ) Drift velocity ( v ) versus voltage ( V ) for ProtK, RNase, and 100 bp dsDNA. Linear fits are used to extract electrophoretic mobility ( μ ) values, as indicated in legend. DNA data are scaled by 0.5×, and dashed line represents resolution limits of our system (see text). To see this figure in color, go online.
    Figure Legend Snippet: ( a and b ) Dwell-time distributions for ProtK ( a ) and RNase ( b ) at selected voltages, along with fits to Eq. 1 with D and v as free parameters ( black line ) and constrained fits for bulk D 0 values ( dashed red line ). Missed regimes shaded red in distributions. ( c ) Diffusion coefficients ( D ) obtained for the proteins from the fits to Eq. 1. ( d ) Drift velocity ( v ) versus voltage ( V ) for ProtK, RNase, and 100 bp dsDNA. Linear fits are used to extract electrophoretic mobility ( μ ) values, as indicated in legend. DNA data are scaled by 0.5×, and dashed line represents resolution limits of our system (see text). To see this figure in color, go online.

    Techniques Used: Diffusion-based Assay

    ( a and b ) Snapshot current traces for RNase ( a ) and ProtK ( b ) at various voltages in the range +200 mV to −200 mV ([KCl] = 1 M, pH 8.1, T = 25°C, low-pass filtered at 125 kHz for presentation only). ( Insets ) Current spikes for V
    Figure Legend Snippet: ( a and b ) Snapshot current traces for RNase ( a ) and ProtK ( b ) at various voltages in the range +200 mV to −200 mV ([KCl] = 1 M, pH 8.1, T = 25°C, low-pass filtered at 125 kHz for presentation only). ( Insets ) Current spikes for V

    Techniques Used:

    Volumetric measurement of proteins using nanopores. ( a ) Fractional current amplitude distributions for RNase and ProtK in an HfO 2 pore at −125 mV. ( b ) Fractional current blockage for the same proteins in an SiN pore ( upper ), and PDB-based cartoons of the proteins and their corresponding Vorlume-based solvent-accessible volumes. When the proteins are mixed at equal stoichiometry, a third peak with a deeper fractional blockage emerges, corresponding to an RNase/ProtK complex ( red , lower ). To see this figure in color, go online.
    Figure Legend Snippet: Volumetric measurement of proteins using nanopores. ( a ) Fractional current amplitude distributions for RNase and ProtK in an HfO 2 pore at −125 mV. ( b ) Fractional current blockage for the same proteins in an SiN pore ( upper ), and PDB-based cartoons of the proteins and their corresponding Vorlume-based solvent-accessible volumes. When the proteins are mixed at equal stoichiometry, a third peak with a deeper fractional blockage emerges, corresponding to an RNase/ProtK complex ( red , lower ). To see this figure in color, go online.

    Techniques Used:

    2) Product Images from "The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans"

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001164

    Mlh1-Mlh3 binding to mismatched and Holliday junction substrates inhibit its endonuclease activity. In native agarose gels, migration of nicked product (n), linear product (black triangle), and closed circular substrate (cc) are indicated. All endonuclease reactions were carried out for 60 min, then stopped by addition of sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and ProteinaseK as described in the Materials and methods. (A) Endonuclease activity was performed with supercoiled pUC18 as described in the Materials and methods. Where + Mg 2+ or + Mn 2+ is indicated, 1 mM MgCl 2 or MnSO 4 was added. Where both Mg 2+ and Mn 2+ are indicated, 0.5 mM of each was included. Where + Mlh1-Mlh3 is indicated, 300 nM wild type or Mlh1-mlh3D523N was added. (B) Mlh1-Mlh3 endonuclease activity on a 2.7 kb circular DNA substrate is inhibited by preincubating Mlh1-Mlh3 with oligonucleotide substrates. Mlh1-Mlh3 (100 nM) was preincubated with increasing amounts of ~50 bp double-stranded oligonucleotide substrates for 15 min at 30°C (0–2,000 nM): either homoduplex (0 μM, 10 μM, 25 μM, 50 μM, 100 μM, or 200 μM, expressed as total nucleotide concentration), +8 loop (0 μM, 10 μM, 25 μM, 50 μM, 100 μM, or 200 μM, expressed as total nucleotide concentration), or 30 bp armed Holliday junction (0 μM, 24 μM, 60 μM, 120 μM, 240 μM, or 480 μM, expressed as total nucleotide concentration). After the preincubation step, reactions were challenged with ~18 μM (expressed as total nucleotide concentration) 2.7 kb circular substrate and incubated by conditions described for endonuclease assays in the Materials and methods and analyzed by agarose gel. All lanes contain 1 mM Mg 2+ . (C) Average of quantification of plasmid nicked for four separate experiments from B; error bars represent standard deviation.
    Figure Legend Snippet: Mlh1-Mlh3 binding to mismatched and Holliday junction substrates inhibit its endonuclease activity. In native agarose gels, migration of nicked product (n), linear product (black triangle), and closed circular substrate (cc) are indicated. All endonuclease reactions were carried out for 60 min, then stopped by addition of sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and ProteinaseK as described in the Materials and methods. (A) Endonuclease activity was performed with supercoiled pUC18 as described in the Materials and methods. Where + Mg 2+ or + Mn 2+ is indicated, 1 mM MgCl 2 or MnSO 4 was added. Where both Mg 2+ and Mn 2+ are indicated, 0.5 mM of each was included. Where + Mlh1-Mlh3 is indicated, 300 nM wild type or Mlh1-mlh3D523N was added. (B) Mlh1-Mlh3 endonuclease activity on a 2.7 kb circular DNA substrate is inhibited by preincubating Mlh1-Mlh3 with oligonucleotide substrates. Mlh1-Mlh3 (100 nM) was preincubated with increasing amounts of ~50 bp double-stranded oligonucleotide substrates for 15 min at 30°C (0–2,000 nM): either homoduplex (0 μM, 10 μM, 25 μM, 50 μM, 100 μM, or 200 μM, expressed as total nucleotide concentration), +8 loop (0 μM, 10 μM, 25 μM, 50 μM, 100 μM, or 200 μM, expressed as total nucleotide concentration), or 30 bp armed Holliday junction (0 μM, 24 μM, 60 μM, 120 μM, 240 μM, or 480 μM, expressed as total nucleotide concentration). After the preincubation step, reactions were challenged with ~18 μM (expressed as total nucleotide concentration) 2.7 kb circular substrate and incubated by conditions described for endonuclease assays in the Materials and methods and analyzed by agarose gel. All lanes contain 1 mM Mg 2+ . (C) Average of quantification of plasmid nicked for four separate experiments from B; error bars represent standard deviation.

    Techniques Used: Binding Assay, Activity Assay, Migration, Concentration Assay, Incubation, Agarose Gel Electrophoresis, Plasmid Preparation, Standard Deviation

    Related Articles

    Incubation:

    Article Title: 5-Formylcytosine mediated DNA–protein cross-links block DNA replication and induce mutations in human cells
    Article Snippet: .. To digest the protein component of DPCs, primer–template complexes containing histone H4-5fC cross-link (2 pmol) were incubated with proteinase K (2.4 units) at 37°C for 48 h in the presence of 1× T4 PNK buffer (New England Biolabs, Beverly, MA, USA). ..

    Article Title: Reprogramming an ATP-driven protein machine into a light-gated nanocage
    Article Snippet: The crosslinking ratio was determined by analysing the samples on a 4–20% sodium dodecyl sulphate (SDS) -PAGE gel (Lonza), and calculating the sum of the relative intensities of the multimer bands (band intensity divided by the sum of the band intensities for all multimers) weighted by their ratio of crosslinks to subunits. .. Proteinase K (New England Biolabs) was added to a final concentration of 20 μg/ml to xMm-cpn samples and samples were incubated for 10min at room temperature before quenching the reaction by addition of 2mM PMSF. .. Ni-NTA-Atto647N (Sigma-Aldrich) or Ni-NTA-nanogold solution (goldiblot kit, Nanoprobes) were added to xMm-cpn and incubated for 30min at room temperature.

    Isolation:

    Article Title: Mitochondrial assembly of the NLRP3 inflammasome complex is initiated at priming
    Article Snippet: Mitochondria were washed, fixed with 4% paraformaldehyde, and analyzed by flow cytometry on a BD LSR Fortessa. .. Protease protection assay was performed on isolated mitochondria by preparing 0.2 mg/ml suspensions of mitochondria and digesting with 20 μg/ml proteinase K (New England Biolabs) with or without 0.5% Triton X-100 for 30 min on ice. .. Following incubation, proteinase K was inactivated with 2 mM phenylmethylsulfonyl fluoride.

    Article Title: Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP)
    Article Snippet: Ligations were performed on-bead (to allow washing away unincorporated adapter) in high concentration of PEG8000, which improves ligation efficiency to > 90%. .. Samples were then run on standard protein gels and transferred to nitrocellulose membranes, and a region 75 kDa (~150 nt of RNA) above the protein size was isolated and proteinase K (NEB) treated to isolate RNA. .. RNA was reverse transcribed with AffinityScript (Agilent), and treated with ExoSAP-IT (Affymetrix) to remove excess oligonucleotides.

    Concentration Assay:

    Article Title: Reprogramming an ATP-driven protein machine into a light-gated nanocage
    Article Snippet: The crosslinking ratio was determined by analysing the samples on a 4–20% sodium dodecyl sulphate (SDS) -PAGE gel (Lonza), and calculating the sum of the relative intensities of the multimer bands (band intensity divided by the sum of the band intensities for all multimers) weighted by their ratio of crosslinks to subunits. .. Proteinase K (New England Biolabs) was added to a final concentration of 20 μg/ml to xMm-cpn samples and samples were incubated for 10min at room temperature before quenching the reaction by addition of 2mM PMSF. .. Ni-NTA-Atto647N (Sigma-Aldrich) or Ni-NTA-nanogold solution (goldiblot kit, Nanoprobes) were added to xMm-cpn and incubated for 30min at room temperature.

    Ethanol Precipitation:

    Article Title: Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes
    Article Snippet: .. Protein was eluted from the beads with 15 μL of 1x SDS loading buffer at 95°C for 2 min. RNA was extracted by treating the beads with 4U proteinase K (NEB) for 2 hrs at 55°C, followed by phenol extraction and ethanol precipitation. .. For some pulldowns, RNase A/T1 mix (EN0551; Fisher Scientific) was applied at 20 U/mg of protein both in the input extract and again while bound to the beads.

    Polymerase Chain Reaction:

    Article Title: TAF-ChIP: An ultra-low input approach for genome wide chromatin immunoprecipitation assay
    Article Snippet: .. The samples were subjected to proteinase K treatment in a 50 μl of TE buffer pH 8.0 with 5 μl of 20 mg/ml of proteinase K. The proteinase K was heat inactivated for 95 °C for 5 min in a 100 μl reaction with 1X NEBNext High-Fidelity PCR Mix, during PCR with primers containing molecular indices (listed in Table 1) with the following program; 72°C for 3min, 95°C for 5min, {98°C for 10 sec, 63°C for 30 sec, 72°C for 30 sec} for 12 cycles, 72°C for 5 min, and hold at 4°C. .. The PCR reaction was purified with bead-based size selection to remove fragments larger than 1000 bp.

    Centrifugation:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: The eIF3-co-purifying complexes were eluted in 50 μl of Elution Buffer (EB – 20 mM Tris–Cl, pH 7.5, 100 mM KCl, 5 mM MgCl2 , 10% glycerol supplemented with the protease inhibitor complete tablet (1 tablet for 12.5 ml of buffer), 1 mM PMSF, 250 mM Imidazole, 0.486 mM β-mercaptoethanol and 2 U of SUPERase In™ RNAse inhibitor for 30 min at 4°C by gentle shaking. .. The resulting eluates were collected by centrifugation at 500 rcf for 2 min (at this step we always preserved 10 μl of eluate as ‘Elute’ for western blot analysis of our routine check-ups of the Ni2+ -pull down efficiency, as described in ( )) and subsequently all proteins in the samples were digested with 0.8 U of Proteinase K (NE Biolabs) at 37°C for 30 min. .. In the case of the eIF2γ Ni2+ -pull down assay, the YMP34 strain was transformed with the selected RaP-NiP constructs along with the GCD11-His allele-carrying vector (pMP65) ( ) and the resulting transformants were cultured in the SD media and subjected to the RaP-NiP as described above.

    Western Blot:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: The eIF3-co-purifying complexes were eluted in 50 μl of Elution Buffer (EB – 20 mM Tris–Cl, pH 7.5, 100 mM KCl, 5 mM MgCl2 , 10% glycerol supplemented with the protease inhibitor complete tablet (1 tablet for 12.5 ml of buffer), 1 mM PMSF, 250 mM Imidazole, 0.486 mM β-mercaptoethanol and 2 U of SUPERase In™ RNAse inhibitor for 30 min at 4°C by gentle shaking. .. The resulting eluates were collected by centrifugation at 500 rcf for 2 min (at this step we always preserved 10 μl of eluate as ‘Elute’ for western blot analysis of our routine check-ups of the Ni2+ -pull down efficiency, as described in ( )) and subsequently all proteins in the samples were digested with 0.8 U of Proteinase K (NE Biolabs) at 37°C for 30 min. .. In the case of the eIF2γ Ni2+ -pull down assay, the YMP34 strain was transformed with the selected RaP-NiP constructs along with the GCD11-His allele-carrying vector (pMP65) ( ) and the resulting transformants were cultured in the SD media and subjected to the RaP-NiP as described above.

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    New England Biolabs proteinase k
    Epithelial cytokine induction is independent of TLR2, TLR4, dectin-1 and MR. Human epithelial cells (1×10 6 ) were pre-incubated with ( A ) 10 µg/ml anti-TLR2, anti-TLR4, anti-MR antibodies, laminarin (100 µg/ml) or S. cerevisiae mannan (40 µg/ml) 2 h before epithelial cells were stimulated with C. albicans walls (1×10 8 ) for 24 h. ( B ) Oral epithelial cells (3×10 5 ) isolated from wild-type and MyD88−/− mice were incubated for 24 h with isolated walls (3×10 7 ). ( C ) Human epithelial cells (1×10 6 ) were incubated with 5 µM cytochalasin D for 30 min prior stimulation with C. albicans walls (1×10 8 ) for 24 h ( D and E ) Cell wall mannoproteins were deproteinized by incubating C. albicans walls (1×10 8 ) with <t>proteinase</t> K or deglycosylated by PNGaseF digestion (cleaves N- glycosylation) or NaOH treatment (alkaline β-elimination reduces O -glycosylation). Human epithelial cells (1×10 6 ) were incubated for 24 h with isolated walls (as positive control) or proteinase K-, PNGaseF- and NaOH-treated walls. ( F and G ) Epithelial cells (1×10 6 ) were incubated for 24 h with cell walls (1×10 8 ) isolated from C. albicans wild type (SC5314), N- glycosylation ( och1Δ ), O -glycosylation ( mnt1Δ/mnt2Δ ), N−/O -glycosylation ( pmr1Δ ) mutant strains or non- pathogenic S. cerevisiae . Human GM-CSF and mouse MIP-2 were quantified by ELISA. TLR4 mRNA up regulation in epithelial cells was determined by quantitative RT-PCR. Data are given as relative mRNA expression compared to mRNA expression of PBS-treated control cells (control = 1.0). ( A–G ), n = 3 (± SEM ), * p
    Proteinase K, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Epithelial cytokine induction is independent of TLR2, TLR4, dectin-1 and MR. Human epithelial cells (1×10 6 ) were pre-incubated with ( A ) 10 µg/ml anti-TLR2, anti-TLR4, anti-MR antibodies, laminarin (100 µg/ml) or S. cerevisiae mannan (40 µg/ml) 2 h before epithelial cells were stimulated with C. albicans walls (1×10 8 ) for 24 h. ( B ) Oral epithelial cells (3×10 5 ) isolated from wild-type and MyD88−/− mice were incubated for 24 h with isolated walls (3×10 7 ). ( C ) Human epithelial cells (1×10 6 ) were incubated with 5 µM cytochalasin D for 30 min prior stimulation with C. albicans walls (1×10 8 ) for 24 h ( D and E ) Cell wall mannoproteins were deproteinized by incubating C. albicans walls (1×10 8 ) with proteinase K or deglycosylated by PNGaseF digestion (cleaves N- glycosylation) or NaOH treatment (alkaline β-elimination reduces O -glycosylation). Human epithelial cells (1×10 6 ) were incubated for 24 h with isolated walls (as positive control) or proteinase K-, PNGaseF- and NaOH-treated walls. ( F and G ) Epithelial cells (1×10 6 ) were incubated for 24 h with cell walls (1×10 8 ) isolated from C. albicans wild type (SC5314), N- glycosylation ( och1Δ ), O -glycosylation ( mnt1Δ/mnt2Δ ), N−/O -glycosylation ( pmr1Δ ) mutant strains or non- pathogenic S. cerevisiae . Human GM-CSF and mouse MIP-2 were quantified by ELISA. TLR4 mRNA up regulation in epithelial cells was determined by quantitative RT-PCR. Data are given as relative mRNA expression compared to mRNA expression of PBS-treated control cells (control = 1.0). ( A–G ), n = 3 (± SEM ), * p

    Journal: PLoS ONE

    Article Title: Glycosylation of Candida albicans Cell Wall Proteins Is Critical for Induction of Innate Immune Responses and Apoptosis of Epithelial Cells

    doi: 10.1371/journal.pone.0050518

    Figure Lengend Snippet: Epithelial cytokine induction is independent of TLR2, TLR4, dectin-1 and MR. Human epithelial cells (1×10 6 ) were pre-incubated with ( A ) 10 µg/ml anti-TLR2, anti-TLR4, anti-MR antibodies, laminarin (100 µg/ml) or S. cerevisiae mannan (40 µg/ml) 2 h before epithelial cells were stimulated with C. albicans walls (1×10 8 ) for 24 h. ( B ) Oral epithelial cells (3×10 5 ) isolated from wild-type and MyD88−/− mice were incubated for 24 h with isolated walls (3×10 7 ). ( C ) Human epithelial cells (1×10 6 ) were incubated with 5 µM cytochalasin D for 30 min prior stimulation with C. albicans walls (1×10 8 ) for 24 h ( D and E ) Cell wall mannoproteins were deproteinized by incubating C. albicans walls (1×10 8 ) with proteinase K or deglycosylated by PNGaseF digestion (cleaves N- glycosylation) or NaOH treatment (alkaline β-elimination reduces O -glycosylation). Human epithelial cells (1×10 6 ) were incubated for 24 h with isolated walls (as positive control) or proteinase K-, PNGaseF- and NaOH-treated walls. ( F and G ) Epithelial cells (1×10 6 ) were incubated for 24 h with cell walls (1×10 8 ) isolated from C. albicans wild type (SC5314), N- glycosylation ( och1Δ ), O -glycosylation ( mnt1Δ/mnt2Δ ), N−/O -glycosylation ( pmr1Δ ) mutant strains or non- pathogenic S. cerevisiae . Human GM-CSF and mouse MIP-2 were quantified by ELISA. TLR4 mRNA up regulation in epithelial cells was determined by quantitative RT-PCR. Data are given as relative mRNA expression compared to mRNA expression of PBS-treated control cells (control = 1.0). ( A–G ), n = 3 (± SEM ), * p

    Article Snippet: Protein degradation was performed by proteinase K (New England BioLabs) digestion (cell wall/proteinase K ratio 50∶1, w/w) for 30 min at 37°C.

    Techniques: Incubation, Isolation, Mouse Assay, Positive Control, Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

    NLRP3 and caspase-1 independently associate with the mitochondria at priming. (A, B) J774A.1 cells were unstimulated, LPS-primed, or LPS-primed followed by stimulation with nigericin (A) or primed with Pam3CSK4 or HMW poly(I:C) (B) . Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (C) J774A.1 cells were LPS-primed for the indicated time in the presence of 10 μM cycloheximide. Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (D) J774A.1 cells were unstimulated or LPS-primed followed by stimulation with nigericin as indicated. Mitochondrial and cytosolic fractions were isolated and mitochondria treated with proteinase K in the presence or absence of Triton X-100. Cytosolic and mitochondrial fractions were then immunoblotted as shown. (E-G) WT, Nlrp3 −/− , Casp1 −/− , and Asc −/− BMM were unstimulated or LPS-primed and stimulated with nigericin as indicated. Mitochondrial and cytosolic fractions were subjected to immunoblot. Data shown are representative of three (A, E, G) or two (B-D, F) independent experiments.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Mitochondrial assembly of the NLRP3 inflammasome complex is initiated at priming

    doi: 10.4049/jimmunol.1701723

    Figure Lengend Snippet: NLRP3 and caspase-1 independently associate with the mitochondria at priming. (A, B) J774A.1 cells were unstimulated, LPS-primed, or LPS-primed followed by stimulation with nigericin (A) or primed with Pam3CSK4 or HMW poly(I:C) (B) . Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (C) J774A.1 cells were LPS-primed for the indicated time in the presence of 10 μM cycloheximide. Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (D) J774A.1 cells were unstimulated or LPS-primed followed by stimulation with nigericin as indicated. Mitochondrial and cytosolic fractions were isolated and mitochondria treated with proteinase K in the presence or absence of Triton X-100. Cytosolic and mitochondrial fractions were then immunoblotted as shown. (E-G) WT, Nlrp3 −/− , Casp1 −/− , and Asc −/− BMM were unstimulated or LPS-primed and stimulated with nigericin as indicated. Mitochondrial and cytosolic fractions were subjected to immunoblot. Data shown are representative of three (A, E, G) or two (B-D, F) independent experiments.

    Article Snippet: Protease protection assay was performed on isolated mitochondria by preparing 0.2 mg/ml suspensions of mitochondria and digesting with 20 μg/ml proteinase K (New England Biolabs) with or without 0.5% Triton X-100 for 30 min on ice.

    Techniques: Isolation

    Schematic overview of TAF-ChIP approach. (1) Formaldehyde fixed cells were directly sorted into RIPA buffer (see methods for details). (2) Cells were briefly sonicated at low intensity to break open the nuclei. (3) Antibodies were coupled to magnetic beads in the presence of blocking reagents. (4) Antibody coupled beads were added to the cell lysate and incubated overnight at 4°C. (5) The tagmentation reaction was performed after initial washes with low salt IP buffer and homemade tagmentation buffer. (6) The tagmentation reaction and the background regions (not anchored by antibody interaction) were washed away with subsequent high stringency washes. (7) The proteinase K was heat-inactivated and the material was PCR-amplified without purification.

    Journal: bioRxiv

    Article Title: TAF-ChIP: An ultra-low input approach for genome wide chromatin immunoprecipitation assay

    doi: 10.1101/299727

    Figure Lengend Snippet: Schematic overview of TAF-ChIP approach. (1) Formaldehyde fixed cells were directly sorted into RIPA buffer (see methods for details). (2) Cells were briefly sonicated at low intensity to break open the nuclei. (3) Antibodies were coupled to magnetic beads in the presence of blocking reagents. (4) Antibody coupled beads were added to the cell lysate and incubated overnight at 4°C. (5) The tagmentation reaction was performed after initial washes with low salt IP buffer and homemade tagmentation buffer. (6) The tagmentation reaction and the background regions (not anchored by antibody interaction) were washed away with subsequent high stringency washes. (7) The proteinase K was heat-inactivated and the material was PCR-amplified without purification.

    Article Snippet: The samples were subjected to proteinase K treatment in a 50 μl of TE buffer pH 8.0 with 5 μl of 20 mg/ml of proteinase K. The proteinase K was heat inactivated for 95 °C for 5 min in a 100 μl reaction with 1X NEBNext High-Fidelity PCR Mix, during PCR with primers containing molecular indices (listed in Table 1) with the following program; 72°C for 3min, 95°C for 5min, {98°C for 10 sec, 63°C for 30 sec, 72°C for 30 sec} for 12 cycles, 72°C for 5 min, and hold at 4°C.

    Techniques: Chromatin Immunoprecipitation, Sonication, Magnetic Beads, Blocking Assay, Incubation, Polymerase Chain Reaction, Amplification, Purification

    Recombinant rREH2 is a catalytically active RNA helicase. (A) Purified rREH2[30–2167] samples in a denaturing gel stained with Coomassie. NTA-Ni elution fractions 1-to-3 are shown with molecular size markers (kDa). (B) Unwinding assays in 10 μL reaction mixtures with increasing concentrations of rREH2[30–2167] +/- ATP. An RNP complex (RNP) forms at the highest enzyme concentration tested; (C) Unwinding assays at increasing incubation times with rREH2[30–2167] +/- ATP using the standard reaction mixture described in the material and methods section. The standard reaction used a molar excess of enzyme over substrate. The dsRNA (ds) was assembled by pre-annealing of synthetic transcript that mimics a 3’ fragment of the T . brucei A6 pre-edited mRNA and a radiolabeled cognate guide RNA [ 27 ]. Radiolabeled unwound ssRNA (ss) is indicated. Input dsRNA was heat denatured (Δ) as control. The assays in this panel were treated with proteinase K to remove any RNP that may accumulate (see methods ). (D) Additional controls showing that the starting radiolabeled ssRNA used to generate the dsRNA substrate in the assay and the unwound radiolabeled ssRNA in the heat-denatured (Δ) dsRNA (in panel C) have the same gel mobility.

    Journal: PLoS ONE

    Article Title: Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes

    doi: 10.1371/journal.pone.0211525

    Figure Lengend Snippet: Recombinant rREH2 is a catalytically active RNA helicase. (A) Purified rREH2[30–2167] samples in a denaturing gel stained with Coomassie. NTA-Ni elution fractions 1-to-3 are shown with molecular size markers (kDa). (B) Unwinding assays in 10 μL reaction mixtures with increasing concentrations of rREH2[30–2167] +/- ATP. An RNP complex (RNP) forms at the highest enzyme concentration tested; (C) Unwinding assays at increasing incubation times with rREH2[30–2167] +/- ATP using the standard reaction mixture described in the material and methods section. The standard reaction used a molar excess of enzyme over substrate. The dsRNA (ds) was assembled by pre-annealing of synthetic transcript that mimics a 3’ fragment of the T . brucei A6 pre-edited mRNA and a radiolabeled cognate guide RNA [ 27 ]. Radiolabeled unwound ssRNA (ss) is indicated. Input dsRNA was heat denatured (Δ) as control. The assays in this panel were treated with proteinase K to remove any RNP that may accumulate (see methods ). (D) Additional controls showing that the starting radiolabeled ssRNA used to generate the dsRNA substrate in the assay and the unwound radiolabeled ssRNA in the heat-denatured (Δ) dsRNA (in panel C) have the same gel mobility.

    Article Snippet: Protein was eluted from the beads with 15 μL of 1x SDS loading buffer at 95°C for 2 min. RNA was extracted by treating the beads with 4U proteinase K (NEB) for 2 hrs at 55°C, followed by phenol extraction and ethanol precipitation.

    Techniques: Recombinant, Purification, Staining, Concentration Assay, Incubation