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Roche buffer
Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 98/100, based on 730 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Centrifugation:

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: .. Nuclei were isolated by centrifugation and resuspended either in SDS-PAGE sample buffer (nucleus fraction) or in 10 mM Tris–HCl (pH 7.4), 0.2 mM MgCl2 , 1% Triton X-100, 5 mM valproic acid, PhosStop (Roche) and Complete protease inhibitors (Roche) for 15 min. Nuclear lysates were centrifuged to isolate the chromatin fraction and supernatant was mixed with SDS-PAGE sample buffer and saved as nucleoplasma fraction. .. The insoluble chromatin fraction was resuspended in SDS-PAGE sample buffer and sonicated to reduce viscosity.

Article Title: Binding of SGTA to Rpn13 selectively modulates protein quality control
Article Snippet: Additional cell culture techniques To purify the proteasomal fraction, parental HEK293T or HEK293Rpn11-HTBH cells were transfected with the indicated plasmids using GeneJuice (Merck) and then lysed 24 h post-transfection in ice-cold buffer (50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 10% (v/v) glycerol, 0.5% (v/v) NP-40) freshly supplemented with complete protease inhibitor cocktail (Roche), 5 mM ATP and 1 mM DTT. .. The resulting lysate was pre-cleared by centrifugation (20 min, 13,000×g , 4°C), and supernatants were incubated with streptavidin beads (Thermo Scientific) at 4°C overnight.

Article Title: The lncRNA Neat1 promotes activation of inflammasomes in macrophages
Article Snippet: Pellets preparation and western blotting Cells were harvested after LPS and nigericin co-treatment, resuspended in 0.3 mL buffer (20 mM Hepes [pH 7.5], 150 mM KCl, 1.5 mM MgCl2 , 1 mM EGTA, 1 mM EDTA, 320 mM sucrose, 1/25 volume of Roche Protease Inhibitor Cocktail, 40 U/mL RNase inhibitor, 20 μM MG132) lysed by shearing 10 times through a 27-gauge syringe. .. After centrifugation (520 × g , 8 min), supernatants were mixed with equal volumes of CHAPS buffer (20 mM Hepes-KOH [pH 7.5], 5 mM MgCl2 , 0.5 mM EGTA, and 0.1% CHAPS) and centrifuged (4000 × g) for another 8 min to pellet the ASC pyroptosomes.

Article Title: Queuosine‐modified tRNAs confer nutritional control of protein translation
Article Snippet: Polysome analysis 107 cells were treated with cycloheximide (100 μg/ml) for 5 minutes washed once in cold PBS/cycloheximide (100 μg/ml) and lysed in 400 μl buffer (20 mM Tris–HCl, pH 7.4, 5 mM MgCl2 , 150 mM NaCl, 1% Triton X‐100, 100 μg/ml cycloheximide, 1× complete protease inhibitors (Roche)). .. Centrifugation was carried out at 35,000 rpm for 2.5 h at 4°C in a Beckmann SW60 rotor.

Amplification:

Article Title: Cloning and Characterization of a Complex DNA Fingerprinting Probe for Candida parapsilosis
Article Snippet: PCRs were performed in 0.5-ml microcentrifuge tubes containing 25 μl of the following reaction mixture: 1 ng of C. parapsilosis DNA, 2.5 μl of 10 × buffer (Roche/Boehringer Mannheim, Indianapolis, Ind.), 1.5 U of Taq DNA polymerase (Roche/Boehringer Mannheim), 200 μM (each) dATP, dCTP, dGTP, and dTTP (Roche/Boehringer Mannheim), and a 0.4 mM concentration of one of the primers listed below. .. The amplification was performed in a thermal cycler (Lab Line, Melrose Park, Ill.) under the following conditions: 45 cycles of 1 min at 94°C, 2 min at 36°C, and 2 min at 73°C.

Isolation:

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: .. Nuclei were isolated by centrifugation and resuspended either in SDS-PAGE sample buffer (nucleus fraction) or in 10 mM Tris–HCl (pH 7.4), 0.2 mM MgCl2 , 1% Triton X-100, 5 mM valproic acid, PhosStop (Roche) and Complete protease inhibitors (Roche) for 15 min. Nuclear lysates were centrifuged to isolate the chromatin fraction and supernatant was mixed with SDS-PAGE sample buffer and saved as nucleoplasma fraction. .. The insoluble chromatin fraction was resuspended in SDS-PAGE sample buffer and sonicated to reduce viscosity.

Article Title: RAV1 Negatively Regulates Seed Development by Directly Repressing MINI3 and IKU2 in Arabidopsis
Article Snippet: Nuclei were isolated in an extraction buffer (1.7 M sucrose, 10 mM Tris-HCl, pH 8.0, 2 mM MgCl2 , 0.15% Trion X-100, 5 mM β-mercaptoethanol, protease inhibitor cocktail (Roche)), and were re-suspended in a sonication buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, protease inhibitor cocktail (Roche)). .. The extracted DNA fragments were diluted with an equal volume of ChIP dilution buffer (16.7 mM Trs-HCl, pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, protease inhibitor cocktail (Roche)) and incubated with polyclonal anti-RAV1 antibody (Ab Frontier) at 4°C overnight.

Blocking Assay:

Article Title: GABAergic neurons in the olfactory cortex projecting to the lateral hypothalamus in mice
Article Snippet: .. After blocking in 1% blocking buffer (11096176001, Roche) for 1 h, the fluorescent-labelled probes were detected. .. The sections were incubated with an anti-fluorescein antibody conjugated with horseradish peroxidase (1:500; Perkin-Elmer) for 1 h at room temperature.

Article Title: Molecular footprinting of skeletal tissues in the catshark Scyliorhinus canicula and the clawed frog Xenopus tropicalis identifies conserved and derived features of vertebrate calcification
Article Snippet: .. In situ hybridization on Scyliorhinus canicula sections DIG-labeled probes were hybridized at 70°C overnight, sections were washed twice in 50% formamide, 1 × SSC, 0.1% Tween-20 for 1 h at 70°C, twice in MABT buffer for 30 min before blocking in blocking buffer (MABT, 2% blocking reagent from Roche, 20% inactivated sheep serum) for 2 h at room temperature. .. Sections were then exposed overnight to a 1:2000 dilution of anti-DIG-AP conjugate antibody (Roche) at 4°C.

Real-time Polymerase Chain Reaction:

Article Title: Similar active genes cluster in specialized transcription factories
Article Snippet: Paragraph title: DNA copy number determined by blotting and qPCR ... Probes were labeled with DIG (digoxigenin) using a DIG labeling kit (Roche) by random priming and hybridized at 65°C for 16 h in “Church” buffer (0.5 M sodium phosphate buffer, 1 mM EDTA, 1% bovine serum albumin, and 7% SDS, pH 7.2), then bound targets were detected using a DIG detection kit (Roche).

Incubation:

Article Title: GABAergic neurons in the olfactory cortex projecting to the lateral hypothalamus in mice
Article Snippet: After blocking in 1% blocking buffer (11096176001, Roche) for 1 h, the fluorescent-labelled probes were detected. .. The sections were incubated with an anti-fluorescein antibody conjugated with horseradish peroxidase (1:500; Perkin-Elmer) for 1 h at room temperature.

Article Title: Molecular footprinting of skeletal tissues in the catshark Scyliorhinus canicula and the clawed frog Xenopus tropicalis identifies conserved and derived features of vertebrate calcification
Article Snippet: In situ hybridization on Scyliorhinus canicula sections DIG-labeled probes were hybridized at 70°C overnight, sections were washed twice in 50% formamide, 1 × SSC, 0.1% Tween-20 for 1 h at 70°C, twice in MABT buffer for 30 min before blocking in blocking buffer (MABT, 2% blocking reagent from Roche, 20% inactivated sheep serum) for 2 h at room temperature. .. After washing, slides were incubated with NBT-BCIP (Roche) staining solution according to the manufacturer's instructions and the reaction stopped by washing in water.

Article Title: De novo transcriptome analyses provide insights into opsin-based photoreception in the lanternshark Etmopterus spinax
Article Snippet: Control sections were incubated in TTBS 5% BSA with no primary antibody. .. Samples (size: 1 cm x 3 cm) were homogenized in 1000 μl of TEN buffer (10 mM Tris, pH 7,5; 1 mM EDTA, pH 8,0; 100 mM NaCl) supplemented with protease inhibitors (complete–Mini tablets, Roche).

Article Title: Nuclear complex of glyceraldehyde-3-phosphate dehydrogenase and DNA repair enzyme apurinic/apyrimidinic endonuclease I protect smooth muscle cells against oxidant-induced cell death
Article Snippet: .. To create double-stranded DNA substrates, equal volumes of 200 µM solutions of the respective and complimentary strands [5′-FAM-AATTCACCGGTACG(F)ACTAGAATTCG-3′ and 5′-CGAATTCTAGTCCGTACCGGTGAATT-3, respectively] were mixed in annealing buffer (50 mM Tris, pH 7.5, 25 mM NaCl, 2 mM MgCl2 , 1 mM DTT, and 0.01% Tween-20) and incubated at 95°C for 5 min. To assay the endonuclease activity of Ape1, cells were harvested in buffer (50 mM Tris-HCL, pH 7.4, 30 mM KCl, and 10% glycerol) that was supplied with protease inhibitors (Complete Ultra tablets; Roche) and lysed by slow shaking at 4°C for 30 min. Ape1 endonuclease reaction mix contained 300 ng cell lysate and 1 μM FAM-labeled Ape1 substrate in endonuclease IV reaction buffer (Thermo Fisher Scientific) that was supplied with 2 mM MgSO4 . .. Mix was incubated at 37°C for 12 min, and reaction was stopped by adding 2× RNA gel loading buffer (Thermo Fisher Scientific).

Article Title: Toll-Like Receptor 9 Deficiency Protects Mice against Pseudomonas aeruginosa Lung Infection
Article Snippet: .. Western Blot After incubation, MHS cells were lysed in buffer [5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 50 mM Tris HCl (pH 7.4)] with anti-proteases (Roche Diagnostics). .. 20 µg of protein lysates were run in a 12% SDS/PAGE, transferred onto PVDF membranes, and probed with antibodies directed against mouse ERK1/2 or its phoshorylated form (Santa Cruz Biotechnology).

Article Title: Binding of SGTA to Rpn13 selectively modulates protein quality control
Article Snippet: Additional cell culture techniques To purify the proteasomal fraction, parental HEK293T or HEK293Rpn11-HTBH cells were transfected with the indicated plasmids using GeneJuice (Merck) and then lysed 24 h post-transfection in ice-cold buffer (50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 10% (v/v) glycerol, 0.5% (v/v) NP-40) freshly supplemented with complete protease inhibitor cocktail (Roche), 5 mM ATP and 1 mM DTT. .. The resulting lysate was pre-cleared by centrifugation (20 min, 13,000×g , 4°C), and supernatants were incubated with streptavidin beads (Thermo Scientific) at 4°C overnight.

Article Title: RAV1 Negatively Regulates Seed Development by Directly Repressing MINI3 and IKU2 in Arabidopsis
Article Snippet: .. The extracted DNA fragments were diluted with an equal volume of ChIP dilution buffer (16.7 mM Trs-HCl, pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, protease inhibitor cocktail (Roche)) and incubated with polyclonal anti-RAV1 antibody (Ab Frontier) at 4°C overnight. .. The chromatin-antibody complex was precipitated with protein-A beads (GE Healthcare) at 4°C for 1 h. Reverse crosslinked DNA was captured and cleaned using the PCR cleanup kit (Favorgen Biotech Corp).

Article Title: The lncRNA Neat1 promotes activation of inflammasomes in macrophages
Article Snippet: Pellets preparation and western blotting Cells were harvested after LPS and nigericin co-treatment, resuspended in 0.3 mL buffer (20 mM Hepes [pH 7.5], 150 mM KCl, 1.5 mM MgCl2 , 1 mM EGTA, 1 mM EDTA, 320 mM sucrose, 1/25 volume of Roche Protease Inhibitor Cocktail, 40 U/mL RNase inhibitor, 20 μM MG132) lysed by shearing 10 times through a 27-gauge syringe. .. The pellets then were resuspended in CHAPS buffer containing 2 mM disuccinimidyl suberate (DSS) and incubated for 30 min followed by centrifugation (4000 × g, 10 min).

Article Title: Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain
Article Snippet: Transport assays Experiments were performed in a 2 mL quartz cuvette with a final volume of 1 mL in a buffer (50 mM Hepes-KOH pH 8.0, 8.5 mM NaCl, 4 mM phosphoenolpyruvate, 60 µg/mL pyruvate kinase (Roche), 2 mM MgCl2 ) and monitored by a Photon Technology International Quanta Master I fluorimeter. .. After incubation at 25 °C during 1 min, inside-out membrane vesicles (IMVs) were added and the fluorescence was recorded.

Activity Assay:

Article Title: Nuclear complex of glyceraldehyde-3-phosphate dehydrogenase and DNA repair enzyme apurinic/apyrimidinic endonuclease I protect smooth muscle cells against oxidant-induced cell death
Article Snippet: .. To create double-stranded DNA substrates, equal volumes of 200 µM solutions of the respective and complimentary strands [5′-FAM-AATTCACCGGTACG(F)ACTAGAATTCG-3′ and 5′-CGAATTCTAGTCCGTACCGGTGAATT-3, respectively] were mixed in annealing buffer (50 mM Tris, pH 7.5, 25 mM NaCl, 2 mM MgCl2 , 1 mM DTT, and 0.01% Tween-20) and incubated at 95°C for 5 min. To assay the endonuclease activity of Ape1, cells were harvested in buffer (50 mM Tris-HCL, pH 7.4, 30 mM KCl, and 10% glycerol) that was supplied with protease inhibitors (Complete Ultra tablets; Roche) and lysed by slow shaking at 4°C for 30 min. Ape1 endonuclease reaction mix contained 300 ng cell lysate and 1 μM FAM-labeled Ape1 substrate in endonuclease IV reaction buffer (Thermo Fisher Scientific) that was supplied with 2 mM MgSO4 . .. Mix was incubated at 37°C for 12 min, and reaction was stopped by adding 2× RNA gel loading buffer (Thermo Fisher Scientific).

BIA-KA:

Article Title: De novo transcriptome analyses provide insights into opsin-based photoreception in the lanternshark Etmopterus spinax
Article Snippet: Samples (size: 1 cm x 3 cm) were homogenized in 1000 μl of TEN buffer (10 mM Tris, pH 7,5; 1 mM EDTA, pH 8,0; 100 mM NaCl) supplemented with protease inhibitors (complete–Mini tablets, Roche). .. Protein concentration in each extract was measured using PierceTM BCA Protein Assay Kit (Thermo Scientific).

Western Blot:

Article Title: Toll-Like Receptor 9 Deficiency Protects Mice against Pseudomonas aeruginosa Lung Infection
Article Snippet: .. Western Blot After incubation, MHS cells were lysed in buffer [5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 50 mM Tris HCl (pH 7.4)] with anti-proteases (Roche Diagnostics). .. 20 µg of protein lysates were run in a 12% SDS/PAGE, transferred onto PVDF membranes, and probed with antibodies directed against mouse ERK1/2 or its phoshorylated form (Santa Cruz Biotechnology).

Article Title: Binding of SGTA to Rpn13 selectively modulates protein quality control
Article Snippet: Additional cell culture techniques To purify the proteasomal fraction, parental HEK293T or HEK293Rpn11-HTBH cells were transfected with the indicated plasmids using GeneJuice (Merck) and then lysed 24 h post-transfection in ice-cold buffer (50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 10% (v/v) glycerol, 0.5% (v/v) NP-40) freshly supplemented with complete protease inhibitor cocktail (Roche), 5 mM ATP and 1 mM DTT. .. The resin was washed with lysis buffer and bound proteins were eluted with SDS sample buffer and resolved by SDS-PAGE followed by western blotting.

Article Title: The lncRNA Neat1 promotes activation of inflammasomes in macrophages
Article Snippet: .. Pellets preparation and western blotting Cells were harvested after LPS and nigericin co-treatment, resuspended in 0.3 mL buffer (20 mM Hepes [pH 7.5], 150 mM KCl, 1.5 mM MgCl2 , 1 mM EGTA, 1 mM EDTA, 320 mM sucrose, 1/25 volume of Roche Protease Inhibitor Cocktail, 40 U/mL RNase inhibitor, 20 μM MG132) lysed by shearing 10 times through a 27-gauge syringe. .. After centrifugation (520 × g , 8 min), supernatants were mixed with equal volumes of CHAPS buffer (20 mM Hepes-KOH [pH 7.5], 5 mM MgCl2 , 0.5 mM EGTA, and 0.1% CHAPS) and centrifuged (4000 × g) for another 8 min to pellet the ASC pyroptosomes.

Hybridization:

Article Title: GABAergic neurons in the olfactory cortex projecting to the lateral hypothalamus in mice
Article Snippet: Hybridization and washing were performed as described above, except that fluorescein-labelled probes were used for hybridization. .. After blocking in 1% blocking buffer (11096176001, Roche) for 1 h, the fluorescent-labelled probes were detected.

Transfection:

Article Title: Binding of SGTA to Rpn13 selectively modulates protein quality control
Article Snippet: .. Additional cell culture techniques To purify the proteasomal fraction, parental HEK293T or HEK293Rpn11-HTBH cells were transfected with the indicated plasmids using GeneJuice (Merck) and then lysed 24 h post-transfection in ice-cold buffer (50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 10% (v/v) glycerol, 0.5% (v/v) NP-40) freshly supplemented with complete protease inhibitor cocktail (Roche), 5 mM ATP and 1 mM DTT. .. The resulting lysate was pre-cleared by centrifugation (20 min, 13,000×g , 4°C), and supernatants were incubated with streptavidin beads (Thermo Scientific) at 4°C overnight.

Immunoprecipitation:

Article Title: Alternative splicing of FBP-interacting repressor coordinates c-Myc, P27Kip1/cyclinE and Ku86/XRCC5 expression as a molecular sensor for bleomycin-induced DNA damage pathway
Article Snippet: Pull-down assays with anti-Ku86 antibody-conjugated beads and protein pathway analysis Nuclear proteins extracted from HLE or HLF cells were immunoprecipitated with anti-Ku86 antibody-conjugated Dynabeads™ (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions [ ]. .. Briefly, cells were homogenized using a Dounce homogenizer in 10 volumes of cold buffer [20 mM HEPES (pH 7.4), 250 mM sucrose, 0.05% NP-40, and protease inhibitor cocktail (Roche Diagnostics)].

Protease Inhibitor:

Article Title: Binding of SGTA to Rpn13 selectively modulates protein quality control
Article Snippet: .. Additional cell culture techniques To purify the proteasomal fraction, parental HEK293T or HEK293Rpn11-HTBH cells were transfected with the indicated plasmids using GeneJuice (Merck) and then lysed 24 h post-transfection in ice-cold buffer (50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 10% (v/v) glycerol, 0.5% (v/v) NP-40) freshly supplemented with complete protease inhibitor cocktail (Roche), 5 mM ATP and 1 mM DTT. .. The resulting lysate was pre-cleared by centrifugation (20 min, 13,000×g , 4°C), and supernatants were incubated with streptavidin beads (Thermo Scientific) at 4°C overnight.

Article Title: RAV1 Negatively Regulates Seed Development by Directly Repressing MINI3 and IKU2 in Arabidopsis
Article Snippet: .. The extracted DNA fragments were diluted with an equal volume of ChIP dilution buffer (16.7 mM Trs-HCl, pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, protease inhibitor cocktail (Roche)) and incubated with polyclonal anti-RAV1 antibody (Ab Frontier) at 4°C overnight. .. The chromatin-antibody complex was precipitated with protein-A beads (GE Healthcare) at 4°C for 1 h. Reverse crosslinked DNA was captured and cleaned using the PCR cleanup kit (Favorgen Biotech Corp).

Article Title: The lncRNA Neat1 promotes activation of inflammasomes in macrophages
Article Snippet: .. Pellets preparation and western blotting Cells were harvested after LPS and nigericin co-treatment, resuspended in 0.3 mL buffer (20 mM Hepes [pH 7.5], 150 mM KCl, 1.5 mM MgCl2 , 1 mM EGTA, 1 mM EDTA, 320 mM sucrose, 1/25 volume of Roche Protease Inhibitor Cocktail, 40 U/mL RNase inhibitor, 20 μM MG132) lysed by shearing 10 times through a 27-gauge syringe. .. After centrifugation (520 × g , 8 min), supernatants were mixed with equal volumes of CHAPS buffer (20 mM Hepes-KOH [pH 7.5], 5 mM MgCl2 , 0.5 mM EGTA, and 0.1% CHAPS) and centrifuged (4000 × g) for another 8 min to pellet the ASC pyroptosomes.

Article Title: Alternative splicing of FBP-interacting repressor coordinates c-Myc, P27Kip1/cyclinE and Ku86/XRCC5 expression as a molecular sensor for bleomycin-induced DNA damage pathway
Article Snippet: .. Briefly, cells were homogenized using a Dounce homogenizer in 10 volumes of cold buffer [20 mM HEPES (pH 7.4), 250 mM sucrose, 0.05% NP-40, and protease inhibitor cocktail (Roche Diagnostics)]. .. The homogenate was centrifuged at 600×g at 4°C for 10 min. After the pellet was washed twice with the same cold buffer, it was solubilized in 10 volumes of RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid sodium salt, 1% NP-40, and protease inhibitor cocktail) and sonicated twice for 10 s. The nuclear fraction (400 μl) was immunoreacted with anti-Ku86 antibody and anti-mouse IgG antibody ( ) conjugated to Dynabeads (100 μl) at 4°C overnight.

Cell Culture:

Article Title: Binding of SGTA to Rpn13 selectively modulates protein quality control
Article Snippet: .. Additional cell culture techniques To purify the proteasomal fraction, parental HEK293T or HEK293Rpn11-HTBH cells were transfected with the indicated plasmids using GeneJuice (Merck) and then lysed 24 h post-transfection in ice-cold buffer (50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 10% (v/v) glycerol, 0.5% (v/v) NP-40) freshly supplemented with complete protease inhibitor cocktail (Roche), 5 mM ATP and 1 mM DTT. .. The resulting lysate was pre-cleared by centrifugation (20 min, 13,000×g , 4°C), and supernatants were incubated with streptavidin beads (Thermo Scientific) at 4°C overnight.

SDS Page:

Article Title: Toll-Like Receptor 9 Deficiency Protects Mice against Pseudomonas aeruginosa Lung Infection
Article Snippet: Western Blot After incubation, MHS cells were lysed in buffer [5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 50 mM Tris HCl (pH 7.4)] with anti-proteases (Roche Diagnostics). .. 20 µg of protein lysates were run in a 12% SDS/PAGE, transferred onto PVDF membranes, and probed with antibodies directed against mouse ERK1/2 or its phoshorylated form (Santa Cruz Biotechnology).

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: .. Nuclei were isolated by centrifugation and resuspended either in SDS-PAGE sample buffer (nucleus fraction) or in 10 mM Tris–HCl (pH 7.4), 0.2 mM MgCl2 , 1% Triton X-100, 5 mM valproic acid, PhosStop (Roche) and Complete protease inhibitors (Roche) for 15 min. Nuclear lysates were centrifuged to isolate the chromatin fraction and supernatant was mixed with SDS-PAGE sample buffer and saved as nucleoplasma fraction. .. The insoluble chromatin fraction was resuspended in SDS-PAGE sample buffer and sonicated to reduce viscosity.

Article Title: Binding of SGTA to Rpn13 selectively modulates protein quality control
Article Snippet: Additional cell culture techniques To purify the proteasomal fraction, parental HEK293T or HEK293Rpn11-HTBH cells were transfected with the indicated plasmids using GeneJuice (Merck) and then lysed 24 h post-transfection in ice-cold buffer (50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 10% (v/v) glycerol, 0.5% (v/v) NP-40) freshly supplemented with complete protease inhibitor cocktail (Roche), 5 mM ATP and 1 mM DTT. .. The resin was washed with lysis buffer and bound proteins were eluted with SDS sample buffer and resolved by SDS-PAGE followed by western blotting.

Article Title: The lncRNA Neat1 promotes activation of inflammasomes in macrophages
Article Snippet: Pellets preparation and western blotting Cells were harvested after LPS and nigericin co-treatment, resuspended in 0.3 mL buffer (20 mM Hepes [pH 7.5], 150 mM KCl, 1.5 mM MgCl2 , 1 mM EGTA, 1 mM EDTA, 320 mM sucrose, 1/25 volume of Roche Protease Inhibitor Cocktail, 40 U/mL RNase inhibitor, 20 μM MG132) lysed by shearing 10 times through a 27-gauge syringe. .. Pellets were fractionated on 12% SDS/PAGE and detected by immunoblotting with ASC antibody.

other:

Article Title: Characterization of the Putative Replisome Organizer of the Lactococcal Bacteriophage r1t
Article Snippet: The pellet was resuspended in 700 μl of sample buffer (50 mM Tris HCl [pH 8.0], 0.3% sodium dodecyl sulfate [SDS], 20 mM dithiothreitol [DTT]), and 7 μl of Prefabloc (Roche Molecular Biochemicals) was added.

Protein Concentration:

Article Title: De novo transcriptome analyses provide insights into opsin-based photoreception in the lanternshark Etmopterus spinax
Article Snippet: Samples (size: 1 cm x 3 cm) were homogenized in 1000 μl of TEN buffer (10 mM Tris, pH 7,5; 1 mM EDTA, pH 8,0; 100 mM NaCl) supplemented with protease inhibitors (complete–Mini tablets, Roche). .. Protein concentration in each extract was measured using PierceTM BCA Protein Assay Kit (Thermo Scientific).

Polymerase Chain Reaction:

Article Title: RAV1 Negatively Regulates Seed Development by Directly Repressing MINI3 and IKU2 in Arabidopsis
Article Snippet: The extracted DNA fragments were diluted with an equal volume of ChIP dilution buffer (16.7 mM Trs-HCl, pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, protease inhibitor cocktail (Roche)) and incubated with polyclonal anti-RAV1 antibody (Ab Frontier) at 4°C overnight. .. The chromatin-antibody complex was precipitated with protein-A beads (GE Healthcare) at 4°C for 1 h. Reverse crosslinked DNA was captured and cleaned using the PCR cleanup kit (Favorgen Biotech Corp).

Article Title: Cloning and Characterization of a Complex DNA Fingerprinting Probe for Candida parapsilosis
Article Snippet: PCRs were performed in 0.5-ml microcentrifuge tubes containing 25 μl of the following reaction mixture: 1 ng of C. parapsilosis DNA, 2.5 μl of 10 × buffer (Roche/Boehringer Mannheim, Indianapolis, Ind.), 1.5 U of Taq DNA polymerase (Roche/Boehringer Mannheim), 200 μM (each) dATP, dCTP, dGTP, and dTTP (Roche/Boehringer Mannheim), and a 0.4 mM concentration of one of the primers listed below. .. PCR products were separated by electrophoresis in a 1.3% (wt/vol) agarose gel and stained with ethidium bromide.

Sonication:

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: Nuclei were isolated by centrifugation and resuspended either in SDS-PAGE sample buffer (nucleus fraction) or in 10 mM Tris–HCl (pH 7.4), 0.2 mM MgCl2 , 1% Triton X-100, 5 mM valproic acid, PhosStop (Roche) and Complete protease inhibitors (Roche) for 15 min. Nuclear lysates were centrifuged to isolate the chromatin fraction and supernatant was mixed with SDS-PAGE sample buffer and saved as nucleoplasma fraction. .. The insoluble chromatin fraction was resuspended in SDS-PAGE sample buffer and sonicated to reduce viscosity.

Article Title: RAV1 Negatively Regulates Seed Development by Directly Repressing MINI3 and IKU2 in Arabidopsis
Article Snippet: The chromatin was released by sonication and DNA fragments with an average size of 0.2–1.5 kb only were recovered. .. The extracted DNA fragments were diluted with an equal volume of ChIP dilution buffer (16.7 mM Trs-HCl, pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, protease inhibitor cocktail (Roche)) and incubated with polyclonal anti-RAV1 antibody (Ab Frontier) at 4°C overnight.

Article Title: Alternative splicing of FBP-interacting repressor coordinates c-Myc, P27Kip1/cyclinE and Ku86/XRCC5 expression as a molecular sensor for bleomycin-induced DNA damage pathway
Article Snippet: Briefly, cells were homogenized using a Dounce homogenizer in 10 volumes of cold buffer [20 mM HEPES (pH 7.4), 250 mM sucrose, 0.05% NP-40, and protease inhibitor cocktail (Roche Diagnostics)]. .. The homogenate was centrifuged at 600×g at 4°C for 10 min. After the pellet was washed twice with the same cold buffer, it was solubilized in 10 volumes of RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid sodium salt, 1% NP-40, and protease inhibitor cocktail) and sonicated twice for 10 s. The nuclear fraction (400 μl) was immunoreacted with anti-Ku86 antibody and anti-mouse IgG antibody ( ) conjugated to Dynabeads (100 μl) at 4°C overnight.

Fluorescence:

Article Title: Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain
Article Snippet: Transport assays Experiments were performed in a 2 mL quartz cuvette with a final volume of 1 mL in a buffer (50 mM Hepes-KOH pH 8.0, 8.5 mM NaCl, 4 mM phosphoenolpyruvate, 60 µg/mL pyruvate kinase (Roche), 2 mM MgCl2 ) and monitored by a Photon Technology International Quanta Master I fluorimeter. .. After incubation at 25 °C during 1 min, inside-out membrane vesicles (IMVs) were added and the fluorescence was recorded.

CtB Assay:

Article Title: GABAergic neurons in the olfactory cortex projecting to the lateral hypothalamus in mice
Article Snippet: For data in Fig. , we performed fluorescent double labelling for CTB immunoreactivity and VGluT1 or GAD65/67 mRNA as follows. .. After blocking in 1% blocking buffer (11096176001, Roche) for 1 h, the fluorescent-labelled probes were detected.

Immunodetection:

Article Title: De novo transcriptome analyses provide insights into opsin-based photoreception in the lanternshark Etmopterus spinax
Article Snippet: Paragraph title: Encephalopsin immunodetection ... Samples (size: 1 cm x 3 cm) were homogenized in 1000 μl of TEN buffer (10 mM Tris, pH 7,5; 1 mM EDTA, pH 8,0; 100 mM NaCl) supplemented with protease inhibitors (complete–Mini tablets, Roche).

Labeling:

Article Title: De novo transcriptome analyses provide insights into opsin-based photoreception in the lanternshark Etmopterus spinax
Article Snippet: Visualization of encephalopsin immunoreactivity was done after a 1 h incubation of the sections at RT with fluorescent dye labeled secondary antibody (Goat Anti-Rabbit, Alexa Fluor 594, Life Technologies Limited) diluted 1:200 in TTBS 5% BSA. .. Samples (size: 1 cm x 3 cm) were homogenized in 1000 μl of TEN buffer (10 mM Tris, pH 7,5; 1 mM EDTA, pH 8,0; 100 mM NaCl) supplemented with protease inhibitors (complete–Mini tablets, Roche).

Article Title: Similar active genes cluster in specialized transcription factories
Article Snippet: .. Probes were labeled with DIG (digoxigenin) using a DIG labeling kit (Roche) by random priming and hybridized at 65°C for 16 h in “Church” buffer (0.5 M sodium phosphate buffer, 1 mM EDTA, 1% bovine serum albumin, and 7% SDS, pH 7.2), then bound targets were detected using a DIG detection kit (Roche). .. Total DNA from ∼107 cells was purified using Trizol (Invitrogen), and the EGFP copy number was determined by qPCR using a thermal cycler (Roter-Gene 3000; Corbett Life Science); signals were normalized relative to those given by GAPDH .

Purification:

Article Title: Similar active genes cluster in specialized transcription factories
Article Snippet: Probes were labeled with DIG (digoxigenin) using a DIG labeling kit (Roche) by random priming and hybridized at 65°C for 16 h in “Church” buffer (0.5 M sodium phosphate buffer, 1 mM EDTA, 1% bovine serum albumin, and 7% SDS, pH 7.2), then bound targets were detected using a DIG detection kit (Roche). .. Total DNA from ∼107 cells was purified using Trizol (Invitrogen), and the EGFP copy number was determined by qPCR using a thermal cycler (Roter-Gene 3000; Corbett Life Science); signals were normalized relative to those given by GAPDH .

Transgenic Assay:

Article Title: RAV1 Negatively Regulates Seed Development by Directly Repressing MINI3 and IKU2 in Arabidopsis
Article Snippet: In brief, 0.8 g of floral tissue at stages 15 to 16 from the RAV1 -overexpressing transgenic plants were cross-linked with 1% formaldehyde for 15 min under a vacuum; the reaction was stopped by adding 0.125 M glycine. .. The extracted DNA fragments were diluted with an equal volume of ChIP dilution buffer (16.7 mM Trs-HCl, pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, protease inhibitor cocktail (Roche)) and incubated with polyclonal anti-RAV1 antibody (Ab Frontier) at 4°C overnight.

Microscopy:

Article Title: De novo transcriptome analyses provide insights into opsin-based photoreception in the lanternshark Etmopterus spinax
Article Snippet: Sections were examined using an epifluorescence microscope (Polyvar SC microscope, Leica Reichter Jung) equipped with a Nikon DS-U1 digital camera coupled with NIS-elements FW software. .. Samples (size: 1 cm x 3 cm) were homogenized in 1000 μl of TEN buffer (10 mM Tris, pH 7,5; 1 mM EDTA, pH 8,0; 100 mM NaCl) supplemented with protease inhibitors (complete–Mini tablets, Roche).

Polyacrylamide Gel Electrophoresis:

Article Title: Nuclear complex of glyceraldehyde-3-phosphate dehydrogenase and DNA repair enzyme apurinic/apyrimidinic endonuclease I protect smooth muscle cells against oxidant-induced cell death
Article Snippet: To create double-stranded DNA substrates, equal volumes of 200 µM solutions of the respective and complimentary strands [5′-FAM-AATTCACCGGTACG(F)ACTAGAATTCG-3′ and 5′-CGAATTCTAGTCCGTACCGGTGAATT-3, respectively] were mixed in annealing buffer (50 mM Tris, pH 7.5, 25 mM NaCl, 2 mM MgCl2 , 1 mM DTT, and 0.01% Tween-20) and incubated at 95°C for 5 min. To assay the endonuclease activity of Ape1, cells were harvested in buffer (50 mM Tris-HCL, pH 7.4, 30 mM KCl, and 10% glycerol) that was supplied with protease inhibitors (Complete Ultra tablets; Roche) and lysed by slow shaking at 4°C for 30 min. Ape1 endonuclease reaction mix contained 300 ng cell lysate and 1 μM FAM-labeled Ape1 substrate in endonuclease IV reaction buffer (Thermo Fisher Scientific) that was supplied with 2 mM MgSO4 . .. Reaction products were separated on 15% PAGE/urea gels and imaged on FLA-7000 phosphoimager (Laser 473 nm, filter 520 nm; FujiFilm, Tokyo, Japan) to quantify the FAM signal.

Staining:

Article Title: Molecular footprinting of skeletal tissues in the catshark Scyliorhinus canicula and the clawed frog Xenopus tropicalis identifies conserved and derived features of vertebrate calcification
Article Snippet: In situ hybridization on Scyliorhinus canicula sections DIG-labeled probes were hybridized at 70°C overnight, sections were washed twice in 50% formamide, 1 × SSC, 0.1% Tween-20 for 1 h at 70°C, twice in MABT buffer for 30 min before blocking in blocking buffer (MABT, 2% blocking reagent from Roche, 20% inactivated sheep serum) for 2 h at room temperature. .. After washing, slides were incubated with NBT-BCIP (Roche) staining solution according to the manufacturer's instructions and the reaction stopped by washing in water.

Article Title: De novo transcriptome analyses provide insights into opsin-based photoreception in the lanternshark Etmopterus spinax
Article Snippet: In order to label the nucleus of each cell, sections have been subject to a DAPI (DAPI nucleic acid stain, Invitrogen) staining during 15 min before being mounted (Mowiol 4–88, Sigma). .. Samples (size: 1 cm x 3 cm) were homogenized in 1000 μl of TEN buffer (10 mM Tris, pH 7,5; 1 mM EDTA, pH 8,0; 100 mM NaCl) supplemented with protease inhibitors (complete–Mini tablets, Roche).

Article Title: Cloning and Characterization of a Complex DNA Fingerprinting Probe for Candida parapsilosis
Article Snippet: PCRs were performed in 0.5-ml microcentrifuge tubes containing 25 μl of the following reaction mixture: 1 ng of C. parapsilosis DNA, 2.5 μl of 10 × buffer (Roche/Boehringer Mannheim, Indianapolis, Ind.), 1.5 U of Taq DNA polymerase (Roche/Boehringer Mannheim), 200 μM (each) dATP, dCTP, dGTP, and dTTP (Roche/Boehringer Mannheim), and a 0.4 mM concentration of one of the primers listed below. .. PCR products were separated by electrophoresis in a 1.3% (wt/vol) agarose gel and stained with ethidium bromide.

Histone Association Assay:

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: Paragraph title: Chromatin association assay ... Nuclei were isolated by centrifugation and resuspended either in SDS-PAGE sample buffer (nucleus fraction) or in 10 mM Tris–HCl (pH 7.4), 0.2 mM MgCl2 , 1% Triton X-100, 5 mM valproic acid, PhosStop (Roche) and Complete protease inhibitors (Roche) for 15 min. Nuclear lysates were centrifuged to isolate the chromatin fraction and supernatant was mixed with SDS-PAGE sample buffer and saved as nucleoplasma fraction.

Chromatin Immunoprecipitation:

Article Title: RAV1 Negatively Regulates Seed Development by Directly Repressing MINI3 and IKU2 in Arabidopsis
Article Snippet: .. The extracted DNA fragments were diluted with an equal volume of ChIP dilution buffer (16.7 mM Trs-HCl, pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, protease inhibitor cocktail (Roche)) and incubated with polyclonal anti-RAV1 antibody (Ab Frontier) at 4°C overnight. .. The chromatin-antibody complex was precipitated with protein-A beads (GE Healthcare) at 4°C for 1 h. Reverse crosslinked DNA was captured and cleaned using the PCR cleanup kit (Favorgen Biotech Corp).

In Situ Hybridization:

Article Title: Molecular footprinting of skeletal tissues in the catshark Scyliorhinus canicula and the clawed frog Xenopus tropicalis identifies conserved and derived features of vertebrate calcification
Article Snippet: .. In situ hybridization on Scyliorhinus canicula sections DIG-labeled probes were hybridized at 70°C overnight, sections were washed twice in 50% formamide, 1 × SSC, 0.1% Tween-20 for 1 h at 70°C, twice in MABT buffer for 30 min before blocking in blocking buffer (MABT, 2% blocking reagent from Roche, 20% inactivated sheep serum) for 2 h at room temperature. .. Sections were then exposed overnight to a 1:2000 dilution of anti-DIG-AP conjugate antibody (Roche) at 4°C.

Plasmid Preparation:

Article Title: Similar active genes cluster in specialized transcription factories
Article Snippet: DNA copy number determined by blotting and qPCR Plasmid DNA from a “Hirt” extract ( ) of ∼107 cells was digested with the appropriate restriction enzyme, separated on an agarose gel, and blotted onto nylon. .. Probes were labeled with DIG (digoxigenin) using a DIG labeling kit (Roche) by random priming and hybridized at 65°C for 16 h in “Church” buffer (0.5 M sodium phosphate buffer, 1 mM EDTA, 1% bovine serum albumin, and 7% SDS, pH 7.2), then bound targets were detected using a DIG detection kit (Roche).

Software:

Article Title: De novo transcriptome analyses provide insights into opsin-based photoreception in the lanternshark Etmopterus spinax
Article Snippet: Sections were examined using an epifluorescence microscope (Polyvar SC microscope, Leica Reichter Jung) equipped with a Nikon DS-U1 digital camera coupled with NIS-elements FW software. .. Samples (size: 1 cm x 3 cm) were homogenized in 1000 μl of TEN buffer (10 mM Tris, pH 7,5; 1 mM EDTA, pH 8,0; 100 mM NaCl) supplemented with protease inhibitors (complete–Mini tablets, Roche).

SYBR Green Assay:

Article Title: Similar active genes cluster in specialized transcription factories
Article Snippet: Probes were labeled with DIG (digoxigenin) using a DIG labeling kit (Roche) by random priming and hybridized at 65°C for 16 h in “Church” buffer (0.5 M sodium phosphate buffer, 1 mM EDTA, 1% bovine serum albumin, and 7% SDS, pH 7.2), then bound targets were detected using a DIG detection kit (Roche). .. In brief, each 25-μl reaction contained Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen) and 5 pmol of each primer (oligo pairs 11 and 12).

Agarose Gel Electrophoresis:

Article Title: Similar active genes cluster in specialized transcription factories
Article Snippet: DNA copy number determined by blotting and qPCR Plasmid DNA from a “Hirt” extract ( ) of ∼107 cells was digested with the appropriate restriction enzyme, separated on an agarose gel, and blotted onto nylon. .. Probes were labeled with DIG (digoxigenin) using a DIG labeling kit (Roche) by random priming and hybridized at 65°C for 16 h in “Church” buffer (0.5 M sodium phosphate buffer, 1 mM EDTA, 1% bovine serum albumin, and 7% SDS, pH 7.2), then bound targets were detected using a DIG detection kit (Roche).

Article Title: Cloning and Characterization of a Complex DNA Fingerprinting Probe for Candida parapsilosis
Article Snippet: PCRs were performed in 0.5-ml microcentrifuge tubes containing 25 μl of the following reaction mixture: 1 ng of C. parapsilosis DNA, 2.5 μl of 10 × buffer (Roche/Boehringer Mannheim, Indianapolis, Ind.), 1.5 U of Taq DNA polymerase (Roche/Boehringer Mannheim), 200 μM (each) dATP, dCTP, dGTP, and dTTP (Roche/Boehringer Mannheim), and a 0.4 mM concentration of one of the primers listed below. .. PCR products were separated by electrophoresis in a 1.3% (wt/vol) agarose gel and stained with ethidium bromide.

In Vitro:

Article Title: Nuclear complex of glyceraldehyde-3-phosphate dehydrogenase and DNA repair enzyme apurinic/apyrimidinic endonuclease I protect smooth muscle cells against oxidant-induced cell death
Article Snippet: The 5′-endonuclease activity of Ape1 was analyzed by using a quantitative in vitro assay that measured the incision of a 26-mer FAM-labeled oligonucleotide substrate that contained a single synthetic AP site [tetrahydrofuran (F)] ( ). .. To create double-stranded DNA substrates, equal volumes of 200 µM solutions of the respective and complimentary strands [5′-FAM-AATTCACCGGTACG(F)ACTAGAATTCG-3′ and 5′-CGAATTCTAGTCCGTACCGGTGAATT-3, respectively] were mixed in annealing buffer (50 mM Tris, pH 7.5, 25 mM NaCl, 2 mM MgCl2 , 1 mM DTT, and 0.01% Tween-20) and incubated at 95°C for 5 min. To assay the endonuclease activity of Ape1, cells were harvested in buffer (50 mM Tris-HCL, pH 7.4, 30 mM KCl, and 10% glycerol) that was supplied with protease inhibitors (Complete Ultra tablets; Roche) and lysed by slow shaking at 4°C for 30 min. Ape1 endonuclease reaction mix contained 300 ng cell lysate and 1 μM FAM-labeled Ape1 substrate in endonuclease IV reaction buffer (Thermo Fisher Scientific) that was supplied with 2 mM MgSO4 .

Electrophoresis:

Article Title: Cloning and Characterization of a Complex DNA Fingerprinting Probe for Candida parapsilosis
Article Snippet: PCRs were performed in 0.5-ml microcentrifuge tubes containing 25 μl of the following reaction mixture: 1 ng of C. parapsilosis DNA, 2.5 μl of 10 × buffer (Roche/Boehringer Mannheim, Indianapolis, Ind.), 1.5 U of Taq DNA polymerase (Roche/Boehringer Mannheim), 200 μM (each) dATP, dCTP, dGTP, and dTTP (Roche/Boehringer Mannheim), and a 0.4 mM concentration of one of the primers listed below. .. PCR products were separated by electrophoresis in a 1.3% (wt/vol) agarose gel and stained with ethidium bromide.

Concentration Assay:

Article Title: Cloning and Characterization of a Complex DNA Fingerprinting Probe for Candida parapsilosis
Article Snippet: .. PCRs were performed in 0.5-ml microcentrifuge tubes containing 25 μl of the following reaction mixture: 1 ng of C. parapsilosis DNA, 2.5 μl of 10 × buffer (Roche/Boehringer Mannheim, Indianapolis, Ind.), 1.5 U of Taq DNA polymerase (Roche/Boehringer Mannheim), 200 μM (each) dATP, dCTP, dGTP, and dTTP (Roche/Boehringer Mannheim), and a 0.4 mM concentration of one of the primers listed below. .. The amplification was performed in a thermal cycler (Lab Line, Melrose Park, Ill.) under the following conditions: 45 cycles of 1 min at 94°C, 2 min at 36°C, and 2 min at 73°C.

In Situ:

Article Title: Molecular footprinting of skeletal tissues in the catshark Scyliorhinus canicula and the clawed frog Xenopus tropicalis identifies conserved and derived features of vertebrate calcification
Article Snippet: In situ hybridization on Scyliorhinus canicula sections DIG-labeled probes were hybridized at 70°C overnight, sections were washed twice in 50% formamide, 1 × SSC, 0.1% Tween-20 for 1 h at 70°C, twice in MABT buffer for 30 min before blocking in blocking buffer (MABT, 2% blocking reagent from Roche, 20% inactivated sheep serum) for 2 h at room temperature. .. Images of in situ hybridizations and histological stainings were taken under a Hamamatsu NanoZoomer 2.0-HT Slide Scanner (40 × objective).

Lysis:

Article Title: Binding of SGTA to Rpn13 selectively modulates protein quality control
Article Snippet: Additional cell culture techniques To purify the proteasomal fraction, parental HEK293T or HEK293Rpn11-HTBH cells were transfected with the indicated plasmids using GeneJuice (Merck) and then lysed 24 h post-transfection in ice-cold buffer (50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 10% (v/v) glycerol, 0.5% (v/v) NP-40) freshly supplemented with complete protease inhibitor cocktail (Roche), 5 mM ATP and 1 mM DTT. .. The resin was washed with lysis buffer and bound proteins were eluted with SDS sample buffer and resolved by SDS-PAGE followed by western blotting.

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  • 96
    Roche immunoprecipitation buffer
    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of <t>co-immunoprecipitation</t> experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).
    Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 228 article reviews
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    95
    Roche flag lysis buffer
    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with <t>anti-FLAG</t> antibodies, resolved by SDS-PAGE, and detected by western blotting using the <t>M2</t> anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.
    Flag Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flag lysis buffer/product/Roche
    Average 95 stars, based on 9 article reviews
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    96
    Roche nonidet p 40 buffer
    Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery. A , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting ( IB ) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8.  Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments.  B , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 ( WT ) or the K273A TRAIL-R1 mutant ( K / A ), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments.  C , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for  panel A. Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments.  D , alignment of primary amino acid sequence of part of the transmembrane segment ( italic ) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in  E . Relevant potential ubiquitination sites are shown in  bold. E , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in  D . TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments.  Asterisk  denotes the heavy chain of the antibody used for IP.
    Nonidet P 40 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche tris buffer
    Localization of TcdE in E. coli and C. difficile . A. Cytoplasmic and membrane proteins analysis of E. coli lysogens of λCmrΔ(SR) carrying pBR322 (control) or pCD463 (+ tcdE -6xHis). B. Cytoplasmic and membrane proteins analysis of C. difficile strain carrying either pMTL84151 (control) or pRG46 (+ tcdE -6xHis). (1) SDS-PAGE coomassie stained gel. Western blots probed with 6XHis Tag antibody (2), ATPase Beta subunit antibody (3), and Ribosomal subunits LI/L2 monoclonal antibody (4). C. Membrane protein samples from bacterial cells expressing TcdE-6His <t>resuspended</t> in denature or native sample buffers and analyzed by Western blot using His-Tag antibody. D. Membrane proteins of JIR8094, TcdE mutant and complemented TcdE mutant strains were harvested from bacterial cultures induced with 20 ng/ml of ATc for 2 hours, separated in 16% <t>Tris-Glycine</t> gel and transferred into PVDF membrane. Panels 1. Ponceau stained membrane; 2. Probed with TcdE antibody.
    Tris Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Journal: Molecular psychiatry

    Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK

    doi: 10.1038/s41380-018-0027-3

    Figure Lengend Snippet: CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C.

    Techniques: Expressing, Construct, Western Blot, Immunoprecipitation, Transfection, Proximity Ligation Assay, Staining, Cell Culture

    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.

    Journal: Journal of viral hepatitis

    Article Title: Sequences in the terminal protein and reverse transcriptase domains of the Hepatitis B Virus polymerase contribute to RNA binding and encapsidation

    doi: 10.1111/jvh.12225

    Figure Lengend Snippet: In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.

    Article Snippet: The FLAG lysis buffer was removed from aliquots of P-bound M2 beads, and then aliquots of beads were incubated with 0.5 μg 32 P-labeled ε RNAs in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris (pH 7.0), 150 mM NaCl, 1 mM EDTA, 0.05% NP-40] with 1× complete protease inhibitor cocktail (Roche), 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 U/μl RNasin Plus RNase inhibitor (Promega) ( ).

    Techniques: In Vitro, RNA Binding Assay, Activity Assay, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Western Blot, Purification, Incubation, Labeling, Binding Assay, Standard Deviation

    Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery. A , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting ( IB ) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8.  Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments.  B , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 ( WT ) or the K273A TRAIL-R1 mutant ( K / A ), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments.  C , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for  panel A. Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments.  D , alignment of primary amino acid sequence of part of the transmembrane segment ( italic ) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in  E . Relevant potential ubiquitination sites are shown in  bold. E , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in  D . TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments.  Asterisk  denotes the heavy chain of the antibody used for IP.

    Journal: The Journal of Biological Chemistry

    Article Title: Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1 *

    doi: 10.1074/jbc.M112.448209

    Figure Lengend Snippet: Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery. A , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting ( IB ) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments. B , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 ( WT ) or the K273A TRAIL-R1 mutant ( K / A ), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments. C , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for panel A. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments. D , alignment of primary amino acid sequence of part of the transmembrane segment ( italic ) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in E . Relevant potential ubiquitination sites are shown in bold. E , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in D . TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments. Asterisk denotes the heavy chain of the antibody used for IP.

    Article Snippet: Western Blotting and Immunoprecipitation Cells were harvested and lysed in Nonidet P-40 buffer consisting of 50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mm NaCl, 1 mm PMSF and Complete Protease Inhibitors (Roche).

    Techniques: Transfection, Mutagenesis, Plasmid Preparation, Isolation, Immunoprecipitation, Sequencing

    Localization of TcdE in E. coli and C. difficile . A. Cytoplasmic and membrane proteins analysis of E. coli lysogens of λCmrΔ(SR) carrying pBR322 (control) or pCD463 (+ tcdE -6xHis). B. Cytoplasmic and membrane proteins analysis of C. difficile strain carrying either pMTL84151 (control) or pRG46 (+ tcdE -6xHis). (1) SDS-PAGE coomassie stained gel. Western blots probed with 6XHis Tag antibody (2), ATPase Beta subunit antibody (3), and Ribosomal subunits LI/L2 monoclonal antibody (4). C. Membrane protein samples from bacterial cells expressing TcdE-6His resuspended in denature or native sample buffers and analyzed by Western blot using His-Tag antibody. D. Membrane proteins of JIR8094, TcdE mutant and complemented TcdE mutant strains were harvested from bacterial cultures induced with 20 ng/ml of ATc for 2 hours, separated in 16% Tris-Glycine gel and transferred into PVDF membrane. Panels 1. Ponceau stained membrane; 2. Probed with TcdE antibody.

    Journal: PLoS Pathogens

    Article Title: Secretion of Clostridium difficile Toxins A and B Requires the Holin-like Protein TcdE

    doi: 10.1371/journal.ppat.1002727

    Figure Lengend Snippet: Localization of TcdE in E. coli and C. difficile . A. Cytoplasmic and membrane proteins analysis of E. coli lysogens of λCmrΔ(SR) carrying pBR322 (control) or pCD463 (+ tcdE -6xHis). B. Cytoplasmic and membrane proteins analysis of C. difficile strain carrying either pMTL84151 (control) or pRG46 (+ tcdE -6xHis). (1) SDS-PAGE coomassie stained gel. Western blots probed with 6XHis Tag antibody (2), ATPase Beta subunit antibody (3), and Ribosomal subunits LI/L2 monoclonal antibody (4). C. Membrane protein samples from bacterial cells expressing TcdE-6His resuspended in denature or native sample buffers and analyzed by Western blot using His-Tag antibody. D. Membrane proteins of JIR8094, TcdE mutant and complemented TcdE mutant strains were harvested from bacterial cultures induced with 20 ng/ml of ATc for 2 hours, separated in 16% Tris-Glycine gel and transferred into PVDF membrane. Panels 1. Ponceau stained membrane; 2. Probed with TcdE antibody.

    Article Snippet: C. difficile toxin and Lactate dehydrogenase (LDH) assays Culture supernatants were collected and filtered, and the cell pellets were resuspended in 10 mM Tris buffer, pH 8.0 containing a protease inhibitor cocktail (Roche, Mannheim, Germany).

    Techniques: SDS Page, Staining, Western Blot, Expressing, Mutagenesis