Structured Review

Roche phenylmethylsulfonyl fluoride pmsf
Phenylmethylsulfonyl Fluoride Pmsf, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 59 article reviews
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phenylmethylsulfonyl fluoride pmsf - by Bioz Stars, 2019-10
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Centrifugation:

Article Title: Aberrant activation of non-coding RNA targets of transcriptional elongation complexes contributes to TDP-43 toxicity
Article Snippet: For human cell samples, cells (~9.6 × 105 ) were lysed in RIPA buffer (Cell Signaling; #9806) supplemented with phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Roche; #1183670001). .. For human cell samples, cells (~9.6 × 105 ) were lysed in RIPA buffer (Cell Signaling; #9806) supplemented with phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Roche; #1183670001).

Article Title: Histone chaperone activity of Arabidopsis thaliana NRP1 is blocked by cytochrome c
Article Snippet: Cell lysates of 25 ml MM2d A. thaliana cultures containing either untreated or 20 μM CPT-treated cells were prepared. .. In both cases, cells were harvested following centrifugation at 3000 × g for 15 min, washed twice in phosphate-buffered saline (PBS), pelleted and resuspended in 0.5 ml buffer containing 20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2 , 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA) and 1 mM dithiothreitol (DTT), supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 1× protease inhibitors (Complete, Roche), 1 mM sodium orthovanadate and 1 mM sodium pyrophosphate. .. Cells were further lysed by sonication (3 cycles 10-s ON, 30-s OFF cycles at 20% amplitude).

Article Title: Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins
Article Snippet: After clearing the lysate by centrifugation for 60 min at 20’000 rpm, 4 °C, the Cglu PafA was purified by nickel affinity chromatography using a 5 ml HisTrap HP column (GE Healthcare). .. The cells were harvested and the pellets were resuspended in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, supplemented with DNAse, Phenylmethylsulfonyl fluoride (PMSF) and Inhibitor Cocktail (Roche) and subsequently lysed by passing the solution four times through an Emulsiflex High Pressure Homogenizer.

Article Title: Changes of Cell Biochemical States Are Revealed in Protein Homomeric Complex Dynamics
Article Snippet: The TAP-tagged LEU1-TAP BY4741 strain (Dharmacon-Horizon Discovery, Cambridge, UK) was grown in 50 mM metformin containing YPD or plain YPD broth until the culture reached an O.D.600 nm of 1.0 (4 replicates for each condition). .. The yeast cells were then harvested by centrifugation, washed and lysed by beadbeating and vortexing 25 times for one minute in lysis buffer (150 mM of NaCl, 50 mM of Tris-HCl pH 8.0, 5 mM of EDTA, 10% (v/v) glycerol, 0.2% (v/v) Nonidet P-40, 1 mM of NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete mini EDTA-free protease inhibitor cocktail from Roche, Mannheim, Germany; ). .. The lysed yeast cell supernatants for both conditions were clarified by centrifugation at 21,000 g for 30 min, and the protein concentrations were determined by a Bradford assay ( ).

Article Title: Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens
Article Snippet: At OD600 =0.75 protein expression was induced with 1 mM isopropyl β-D -1-thiogalactopyranoside (IPTG), and the cells grown overnight at 20 °C. .. Cells were harvested by centrifugation, and cell pellets dissolved in 10 ml fusion protein lysis buffer (50 mM Tris, 25 mM NaCl, 5% glycerol, pH 7.8) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail mixture (Roche) per litre of starting culture. .. Lysozyme (Merck) was added to a final concentration of 1 mg ml−1 and the cell suspension was incubated for 1 h on ice.

Filtration:

Article Title: Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins
Article Snippet: Size exclusion chromatography was carried out on a Superose 6 pg gel filtration column equilibrated and run in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, 1 mM DTT, 1 mM EDTA and 10% glycerol. .. The cells were harvested and the pellets were resuspended in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, supplemented with DNAse, Phenylmethylsulfonyl fluoride (PMSF) and Inhibitor Cocktail (Roche) and subsequently lysed by passing the solution four times through an Emulsiflex High Pressure Homogenizer.

Cycling Probe Technology:

Article Title: Histone chaperone activity of Arabidopsis thaliana NRP1 is blocked by cytochrome c
Article Snippet: Cell lysates of 25 ml MM2d A. thaliana cultures containing either untreated or 20 μM CPT-treated cells were prepared. .. In both cases, cells were harvested following centrifugation at 3000 × g for 15 min, washed twice in phosphate-buffered saline (PBS), pelleted and resuspended in 0.5 ml buffer containing 20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2 , 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA) and 1 mM dithiothreitol (DTT), supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 1× protease inhibitors (Complete, Roche), 1 mM sodium orthovanadate and 1 mM sodium pyrophosphate.

Stable Transfection:

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability
Article Snippet: HN30 cells were cultured in DMEM medium supplemented with 10% FBS. .. To detect BirA*-RASG12V proteins and biotin labeling, HEK-HT cells or HEK-HT cells stably infected with LentiCRISPR2.0 or LentiCRISPR2.0-sgPIP5K1A-1, -2, or -3 were transiently transfected with pcDNA3.1-myc-BirA*-KRASG12V , -NRASG12V , and/or -HRASG12V were treated with biotin (see the Biotin identification section below) and then lysed in buffer containing 1% NP-40, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Roche) by end-to-end incubation at 4 °C for 30 min. Lysates were centrifuged at 21,000 x g for 10 min. Total protein was quantified using BCA kit (Thermo Fisher Scientific). .. Proteins were separated on 12.5% or gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad) and transferred onto polyvinylidene difluoride membranes and probed with anti-myc (Cell Signaling, #2276; 1:1000) or β-actin (Cell Signaling #3700; 1:5000) primary antibodies in blocking buffer containing either 5% milk or 5% bovine serum albumin followed by the secondary antibodies of goat anti-rabbit IgG (H+L) HRP (Thermo Fisher Scientific, #65-6120, 1:2000) or goat anti-mouse IgG (H+L) HRP (Life Technologies, #G21040, 1:5000).

Construct:

Article Title: Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE
Article Snippet: The untagged TWINKLE was purified using N-His6 -SUMO-fused TWINKLE construct made using Champion pET SUMO expression system from Invitrogen and Escherichia coli Rosetta (DE3) cells. .. Briefly, cells were lysed using lysozyme (0.2 mg/ml) and repeated freeze-thaw cycles in liquid nitrogen after pellet resuspension in Buffer A (50 mM Tris Cl pH 7.1, 600 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% Glycerol, 0.1% Tween, 0.2 mM 1.4-Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl fluoride (PMSF) and Roche protease inhibitor tablets).

Article Title: Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens
Article Snippet: Paragraph title: Expression and purification of TXNDC11 constructs containing the coiled-coil domain ... Cells were harvested by centrifugation, and cell pellets dissolved in 10 ml fusion protein lysis buffer (50 mM Tris, 25 mM NaCl, 5% glycerol, pH 7.8) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail mixture (Roche) per litre of starting culture.

SYBR Green Assay:

Article Title: Identification and characterization of the role of c-terminal Src kinase in dengue virus replication
Article Snippet: Real time PCRs were performed by using one-step or two-step methods using SYBR green or Taqman chemistry as described previously . .. For immunoblotting, cell lysates were prepared in lysis buffer (50 mM Tris-Cl pH-8.0, 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS) containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (PIC) (Roche).

Incubation:

Article Title: The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition
Article Snippet: The pellet was re-suspended in buffer A (50 mM Tris–HCl pH8.0, 300 mM NaCl) containing 2 mM phenylmethanesulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablets (Roche). .. Cells were lysed by sonication and the cell debris were removed by centrifugation at 30 000 g for 45 min at 4°C.

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability
Article Snippet: HN30 cells were cultured in DMEM medium supplemented with 10% FBS. .. To detect BirA*-RASG12V proteins and biotin labeling, HEK-HT cells or HEK-HT cells stably infected with LentiCRISPR2.0 or LentiCRISPR2.0-sgPIP5K1A-1, -2, or -3 were transiently transfected with pcDNA3.1-myc-BirA*-KRASG12V , -NRASG12V , and/or -HRASG12V were treated with biotin (see the Biotin identification section below) and then lysed in buffer containing 1% NP-40, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Roche) by end-to-end incubation at 4 °C for 30 min. Lysates were centrifuged at 21,000 x g for 10 min. Total protein was quantified using BCA kit (Thermo Fisher Scientific). .. Proteins were separated on 12.5% or gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad) and transferred onto polyvinylidene difluoride membranes and probed with anti-myc (Cell Signaling, #2276; 1:1000) or β-actin (Cell Signaling #3700; 1:5000) primary antibodies in blocking buffer containing either 5% milk or 5% bovine serum albumin followed by the secondary antibodies of goat anti-rabbit IgG (H+L) HRP (Thermo Fisher Scientific, #65-6120, 1:2000) or goat anti-mouse IgG (H+L) HRP (Life Technologies, #G21040, 1:5000).

Article Title: A novel HDAC inhibitor, CG200745, inhibits pancreatic cancer cell growth and overcomes gemcitabine resistance
Article Snippet: Cells were lysed in a lysis buffer containing 70 mM glycerophosphate (pH 7.2), 0.6 mM Na vanadate, 2 mM MgCl2 , 1 mM EGTA, 1 mM DTT, 0.5% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 1× complete protease inhibitor (Roche Applied Science, Nutley, NJ, USA). .. Cells were lysed in a lysis buffer containing 70 mM glycerophosphate (pH 7.2), 0.6 mM Na vanadate, 2 mM MgCl2 , 1 mM EGTA, 1 mM DTT, 0.5% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 1× complete protease inhibitor (Roche Applied Science, Nutley, NJ, USA).

Article Title: Rad51 Interacts with Non-structural 3 Protein of Hepatitis C Virus and Regulates Viral Production
Article Snippet: Cells were harvested and lysed in buffer containing 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.25% sodium deoxycholate, 1 mM Na3 VO4 , 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM β-glycerophosphate, and protease inhibitor cocktail (Roche) for 30 min on ice, and centrifuged at 12,000 rpm for 10 min at 4°C. .. The supernatant was collected and equal amounts of protein were subjected to SDS-PAGE and electrotransferred to a nitrocellulose membrane.

Article Title: Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens
Article Snippet: Cells were harvested by centrifugation, and cell pellets dissolved in 10 ml fusion protein lysis buffer (50 mM Tris, 25 mM NaCl, 5% glycerol, pH 7.8) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail mixture (Roche) per litre of starting culture. .. For Ni-NTA purification, the cleared lysate containing co-expressed MBP-Trx5-CC C743S/C788S and Ub-His-Trx5-CC C743S/C788S was filtered and diluted with an equal volume of Ni-NTA wash buffer (50 mM Tris, 20 mM imidazole, 250 mM NaCl, 1% glycerol, pH 7.8).

Expressing:

Article Title: The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition
Article Snippet: Paragraph title: Expression and purification of recombinant proteins ... The pellet was re-suspended in buffer A (50 mM Tris–HCl pH8.0, 300 mM NaCl) containing 2 mM phenylmethanesulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablets (Roche).

Article Title: Source of the Fitness Defect in Rifamycin-Resistant Mycobacterium tuberculosis RNA Polymerase and the Mechanism of Compensation by Mutations in the β′ Subunit
Article Snippet: Protein expression was induced by addition of 1 mM IPTG (isopropyl-β- d -thiogalactopyranoside). .. Cells were lysed via sonication in 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol (β-ME), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1× Roche cOmplete ULTRA protease cocktail, 200 U of DNase I, and 1 mg/ml lysozyme.

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability
Article Snippet: To detect BirA*-RASG12V proteins and biotin labeling, HEK-HT cells or HEK-HT cells stably infected with LentiCRISPR2.0 or LentiCRISPR2.0-sgPIP5K1A-1, -2, or -3 were transiently transfected with pcDNA3.1-myc-BirA*-KRASG12V , -NRASG12V , and/or -HRASG12V were treated with biotin (see the Biotin identification section below) and then lysed in buffer containing 1% NP-40, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Roche) by end-to-end incubation at 4 °C for 30 min. Lysates were centrifuged at 21,000 x g for 10 min. Total protein was quantified using BCA kit (Thermo Fisher Scientific). .. To detect BirA*-RASG12V proteins and biotin labeling, HEK-HT cells or HEK-HT cells stably infected with LentiCRISPR2.0 or LentiCRISPR2.0-sgPIP5K1A-1, -2, or -3 were transiently transfected with pcDNA3.1-myc-BirA*-KRASG12V , -NRASG12V , and/or -HRASG12V were treated with biotin (see the Biotin identification section below) and then lysed in buffer containing 1% NP-40, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Roche) by end-to-end incubation at 4 °C for 30 min. Lysates were centrifuged at 21,000 x g for 10 min. Total protein was quantified using BCA kit (Thermo Fisher Scientific).

Article Title: Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE
Article Snippet: The untagged TWINKLE was purified using N-His6 -SUMO-fused TWINKLE construct made using Champion pET SUMO expression system from Invitrogen and Escherichia coli Rosetta (DE3) cells. .. Briefly, cells were lysed using lysozyme (0.2 mg/ml) and repeated freeze-thaw cycles in liquid nitrogen after pellet resuspension in Buffer A (50 mM Tris Cl pH 7.1, 600 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% Glycerol, 0.1% Tween, 0.2 mM 1.4-Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl fluoride (PMSF) and Roche protease inhibitor tablets).

Article Title: Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation
Article Snippet: Paragraph title: Expression and Purification of Recombinant hDPCK in Bacteria ... Cells were lysed by a French press in 50 mM Tris-HCl (pH 8), 0.5 M NaCl, 1% Triton X-100, 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM β-mercaptoethanol, and Roche Complete EDTA-free protease inhibitor cocktail.

Article Title: Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins
Article Snippet: Paragraph title: Protein expression ... The cells were harvested and the pellets were resuspended in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, supplemented with DNAse, Phenylmethylsulfonyl fluoride (PMSF) and Inhibitor Cocktail (Roche) and subsequently lysed by passing the solution four times through an Emulsiflex High Pressure Homogenizer.

Article Title: Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens
Article Snippet: Paragraph title: Expression and purification of TXNDC11 constructs containing the coiled-coil domain ... Cells were harvested by centrifugation, and cell pellets dissolved in 10 ml fusion protein lysis buffer (50 mM Tris, 25 mM NaCl, 5% glycerol, pH 7.8) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail mixture (Roche) per litre of starting culture.

BIA-KA:

Article Title: Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain
Article Snippet: Frozen tissue was homogenized (0.5–1 mg mL−1 ) in ice-cold lysis buffer containing 50 mM Tris-buffered saline (TBS, pH 7.4), 0.1 % (v/v) Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM ethylene glycol tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete™ protease inhibitor (Roche Diagnostics Ltd., West Sussex, UK). .. Frozen tissue was homogenized (0.5–1 mg mL−1 ) in ice-cold lysis buffer containing 50 mM Tris-buffered saline (TBS, pH 7.4), 0.1 % (v/v) Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM ethylene glycol tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete™ protease inhibitor (Roche Diagnostics Ltd., West Sussex, UK).

Article Title: Aberrant activation of non-coding RNA targets of transcriptional elongation complexes contributes to TDP-43 toxicity
Article Snippet: For human cell samples, cells (~9.6 × 105 ) were lysed in RIPA buffer (Cell Signaling; #9806) supplemented with phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Roche; #1183670001). .. For human cell samples, cells (~9.6 × 105 ) were lysed in RIPA buffer (Cell Signaling; #9806) supplemented with phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Roche; #1183670001).

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability
Article Snippet: HN30 cells were cultured in DMEM medium supplemented with 10% FBS. .. To detect BirA*-RASG12V proteins and biotin labeling, HEK-HT cells or HEK-HT cells stably infected with LentiCRISPR2.0 or LentiCRISPR2.0-sgPIP5K1A-1, -2, or -3 were transiently transfected with pcDNA3.1-myc-BirA*-KRASG12V , -NRASG12V , and/or -HRASG12V were treated with biotin (see the Biotin identification section below) and then lysed in buffer containing 1% NP-40, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Roche) by end-to-end incubation at 4 °C for 30 min. Lysates were centrifuged at 21,000 x g for 10 min. Total protein was quantified using BCA kit (Thermo Fisher Scientific). .. Proteins were separated on 12.5% or gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad) and transferred onto polyvinylidene difluoride membranes and probed with anti-myc (Cell Signaling, #2276; 1:1000) or β-actin (Cell Signaling #3700; 1:5000) primary antibodies in blocking buffer containing either 5% milk or 5% bovine serum albumin followed by the secondary antibodies of goat anti-rabbit IgG (H+L) HRP (Thermo Fisher Scientific, #65-6120, 1:2000) or goat anti-mouse IgG (H+L) HRP (Life Technologies, #G21040, 1:5000).

Western Blot:

Article Title: Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain
Article Snippet: Frozen tissue was homogenized (0.5–1 mg mL−1 ) in ice-cold lysis buffer containing 50 mM Tris-buffered saline (TBS, pH 7.4), 0.1 % (v/v) Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM ethylene glycol tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete™ protease inhibitor (Roche Diagnostics Ltd., West Sussex, UK). .. Protein concentrations in supernatants were measured using a BCA protein assay kit (Pierce Endogen, Rockford, USA).

Article Title: Aberrant activation of non-coding RNA targets of transcriptional elongation complexes contributes to TDP-43 toxicity
Article Snippet: Paragraph title: Western blotting ... For human cell samples, cells (~9.6 × 105 ) were lysed in RIPA buffer (Cell Signaling; #9806) supplemented with phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Roche; #1183670001).

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability
Article Snippet: To detect BirA*-RASG12V proteins and biotin labeling, HEK-HT cells or HEK-HT cells stably infected with LentiCRISPR2.0 or LentiCRISPR2.0-sgPIP5K1A-1, -2, or -3 were transiently transfected with pcDNA3.1-myc-BirA*-KRASG12V , -NRASG12V , and/or -HRASG12V were treated with biotin (see the Biotin identification section below) and then lysed in buffer containing 1% NP-40, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Roche) by end-to-end incubation at 4 °C for 30 min. Lysates were centrifuged at 21,000 x g for 10 min. Total protein was quantified using BCA kit (Thermo Fisher Scientific). .. Proteins were separated on 12.5% or gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad) and transferred onto polyvinylidene difluoride membranes and probed with anti-myc (Cell Signaling, #2276; 1:1000) or β-actin (Cell Signaling #3700; 1:5000) primary antibodies in blocking buffer containing either 5% milk or 5% bovine serum albumin followed by the secondary antibodies of goat anti-rabbit IgG (H+L) HRP (Thermo Fisher Scientific, #65-6120, 1:2000) or goat anti-mouse IgG (H+L) HRP (Life Technologies, #G21040, 1:5000).

Article Title: A novel HDAC inhibitor, CG200745, inhibits pancreatic cancer cell growth and overcomes gemcitabine resistance
Article Snippet: Paragraph title: Western blotting ... Cells were lysed in a lysis buffer containing 70 mM glycerophosphate (pH 7.2), 0.6 mM Na vanadate, 2 mM MgCl2 , 1 mM EGTA, 1 mM DTT, 0.5% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 1× complete protease inhibitor (Roche Applied Science, Nutley, NJ, USA).

Article Title: Identification and characterization of the role of c-terminal Src kinase in dengue virus replication
Article Snippet: Paragraph title: Viral titers, real time PCR and western blotting ... For immunoblotting, cell lysates were prepared in lysis buffer (50 mM Tris-Cl pH-8.0, 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS) containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (PIC) (Roche).

Article Title: Efficacy of EZH2 inhibitory drugs in human papillomavirus-positive and human papillomavirus-negative oropharyngeal squamous cell carcinomas
Article Snippet: Paragraph title: Whole cell lysate and Western blot ... Whole cell lysates were utilized in the analysis of EZH2 protein content and were obtained from harvesting cells in 200 μL 50 mM TRIS +1% SDS pH 7.6, supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 × complete protease inhibitor (CPI) (Cat#: 11697498001 Roche).

Article Title: Differential PKA activation and AKAP association determines cell fate in cancer cells
Article Snippet: Paragraph title: Antibodies, western blotting and immunoprecipitation ... Cells were lysed in TNESV lysis buffer (50 mmol/liter Tris (pH 7.5), 150 mmol/liter NaCl, 1% NP40, 50 mmol/liter NaF, 1 mmol/liter Na3VO4, 25 μg/ml β-glycerophosphate, 1 mmol/liter phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Roche, Indianapolis, IN) as previously described [ , , , ].

Article Title: Acidified bile acids enhance tumor progression and telomerase activity of gastric cancer in mice dependent on c‐Myc expression
Article Snippet: Paragraph title: Western blot analysis ... All protein samples were homogenized on ice in lysis buffer containing 40 mmol/L Tris‐HCl (pH 6.9), 2 mmol/L ethylenediaminetetraacetic acid (EDTA, pH 8.0), 100 mmol/L sodium fluoride, 150 mmol/L NaCl, 10 mmol/L sodium pyrophosphate, 1% Tergitol type NP‐40, 2 mmol/L sodium orthovanadate, 1% Triton X‐100, 1.0 mmol/L phenylmethanesulfonyl fluoride (PMSF), and 1 × protein inhibitor mini‐tablet (Roche, Basel, Switzerland).

Article Title: Clinically used antirheumatic agent auranofin is a proteasomal deubiquitinase inhibitor and inhibits tumor growth
Article Snippet: Paragraph title: Western blot analysis ... Whole cell lysates were prepared in RIPA buffer supplemented with 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1×Roche Complete Mini Protease Inhibitor Cocktail (Roche, Indianapolis, IN).

Crystallization Assay:

Article Title: The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition
Article Snippet: The pellet was re-suspended in buffer A (50 mM Tris–HCl pH8.0, 300 mM NaCl) containing 2 mM phenylmethanesulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablets (Roche). .. The protein was further purified by size-exclusion chromatography using Sephacryl S200 Hi-Prep 16/60 column (GE Healthcare, Inc .).

Transformation Assay:

Article Title: The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition
Article Snippet: In each case, the plasmid with the gene of interest was transformed into Escherichia coli BL21(DE3) strain (Novagen Inc .). .. The pellet was re-suspended in buffer A (50 mM Tris–HCl pH8.0, 300 mM NaCl) containing 2 mM phenylmethanesulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablets (Roche).

Article Title: Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins
Article Snippet: The cells were harvested and the pellets were resuspended in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, supplemented with DNAse, Phenylmethylsulfonyl fluoride (PMSF) and Inhibitor Cocktail (Roche) and subsequently lysed by passing the solution four times through an Emulsiflex High Pressure Homogenizer. .. To remove uncleaved protein, the cleaved hexahistidine tag and the TEV protease, the protein solution was again passed over the 5 ml HisTrap HP column and further purified using a Superose 6 pg in buffer containing 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, 1 mM DTT, 1 mM EDTA and 10% glycerol.

Article Title: Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens
Article Snippet: Plasmids pLE523 and/or pLE525 were transformed into competent BL21(DE3) cells and plated onto LB plates containing the appropriate antibiotic(s) (ampicillin (100 μg ml−1 ) and/or kanamycin (50 μg m−1 l)). .. Cells were harvested by centrifugation, and cell pellets dissolved in 10 ml fusion protein lysis buffer (50 mM Tris, 25 mM NaCl, 5% glycerol, pH 7.8) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail mixture (Roche) per litre of starting culture.

Derivative Assay:

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability
Article Snippet: To detect BirA*-RASG12V proteins and biotin labeling, HEK-HT cells or HEK-HT cells stably infected with LentiCRISPR2.0 or LentiCRISPR2.0-sgPIP5K1A-1, -2, or -3 were transiently transfected with pcDNA3.1-myc-BirA*-KRASG12V , -NRASG12V , and/or -HRASG12V were treated with biotin (see the Biotin identification section below) and then lysed in buffer containing 1% NP-40, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Roche) by end-to-end incubation at 4 °C for 30 min. Lysates were centrifuged at 21,000 x g for 10 min. Total protein was quantified using BCA kit (Thermo Fisher Scientific). .. To detect BirA*-RASG12V proteins and biotin labeling, HEK-HT cells or HEK-HT cells stably infected with LentiCRISPR2.0 or LentiCRISPR2.0-sgPIP5K1A-1, -2, or -3 were transiently transfected with pcDNA3.1-myc-BirA*-KRASG12V , -NRASG12V , and/or -HRASG12V were treated with biotin (see the Biotin identification section below) and then lysed in buffer containing 1% NP-40, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Roche) by end-to-end incubation at 4 °C for 30 min. Lysates were centrifuged at 21,000 x g for 10 min. Total protein was quantified using BCA kit (Thermo Fisher Scientific).

Flow Cytometry:

Article Title: Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE
Article Snippet: Briefly, cells were lysed using lysozyme (0.2 mg/ml) and repeated freeze-thaw cycles in liquid nitrogen after pellet resuspension in Buffer A (50 mM Tris Cl pH 7.1, 600 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% Glycerol, 0.1% Tween, 0.2 mM 1.4-Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl fluoride (PMSF) and Roche protease inhibitor tablets). .. Briefly, cells were lysed using lysozyme (0.2 mg/ml) and repeated freeze-thaw cycles in liquid nitrogen after pellet resuspension in Buffer A (50 mM Tris Cl pH 7.1, 600 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% Glycerol, 0.1% Tween, 0.2 mM 1.4-Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl fluoride (PMSF) and Roche protease inhibitor tablets).

Concentration Assay:

Article Title: Changes of Cell Biochemical States Are Revealed in Protein Homomeric Complex Dynamics
Article Snippet: Paragraph title: Analysis of concentration of protein-bound iron by ICP-MS ... The yeast cells were then harvested by centrifugation, washed and lysed by beadbeating and vortexing 25 times for one minute in lysis buffer (150 mM of NaCl, 50 mM of Tris-HCl pH 8.0, 5 mM of EDTA, 10% (v/v) glycerol, 0.2% (v/v) Nonidet P-40, 1 mM of NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete mini EDTA-free protease inhibitor cocktail from Roche, Mannheim, Germany; ).

Article Title: Differential PKA activation and AKAP association determines cell fate in cancer cells
Article Snippet: Cells were lysed in TNESV lysis buffer (50 mmol/liter Tris (pH 7.5), 150 mmol/liter NaCl, 1% NP40, 50 mmol/liter NaF, 1 mmol/liter Na3VO4, 25 μg/ml β-glycerophosphate, 1 mmol/liter phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Roche, Indianapolis, IN) as previously described [ , , , ]. .. Co- Immunoprecipitation (IP) was performed with 500 μg protein aliquots using magnetic beads (Millipore) according to the instructions provided by the manufacturer (# LSKMAGAG10) and previously established method [ ].

Protease Inhibitor:

Article Title: Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain
Article Snippet: Frozen postmortem human temporal cortex (Table 1) from control (n = 5) and pathologically confirmed cases of sporadic AD of Braak stage II (n = 5), III (3), IV (n = 4), V (n = 3) and VI (n = 5) were obtained from the MRC London Neurodegenerative Diseases Brain Bank (Additional file : Table S1, Additional file : Table S2). .. Frozen tissue was homogenized (0.5–1 mg mL−1 ) in ice-cold lysis buffer containing 50 mM Tris-buffered saline (TBS, pH 7.4), 0.1 % (v/v) Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM ethylene glycol tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete™ protease inhibitor (Roche Diagnostics Ltd., West Sussex, UK). .. Homogenates were centrifuged at 25,000g(av) for 20 min at 4 °C.

Article Title: The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition
Article Snippet: Post-induction, cultures were grown for 12 h at 18°C. .. The pellet was re-suspended in buffer A (50 mM Tris–HCl pH8.0, 300 mM NaCl) containing 2 mM phenylmethanesulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablets (Roche). .. Cells were lysed by sonication and the cell debris were removed by centrifugation at 30 000 g for 45 min at 4°C.

Article Title: Aberrant activation of non-coding RNA targets of transcriptional elongation complexes contributes to TDP-43 toxicity
Article Snippet: Proteins were transferred to nitrocellulose membrane by the iBlot blotting system (ThermoFisher). .. For human cell samples, cells (~9.6 × 105 ) were lysed in RIPA buffer (Cell Signaling; #9806) supplemented with phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Roche; #1183670001). .. Extracts were sonicated for 6 min using using QSonica (Newtown, CT) water bath sonicator (amplitude = 100, 30-s on, 30-s off).

Article Title: Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE
Article Snippet: The untagged TWINKLE was purified using N-His6 -SUMO-fused TWINKLE construct made using Champion pET SUMO expression system from Invitrogen and Escherichia coli Rosetta (DE3) cells. .. Briefly, cells were lysed using lysozyme (0.2 mg/ml) and repeated freeze-thaw cycles in liquid nitrogen after pellet resuspension in Buffer A (50 mM Tris Cl pH 7.1, 600 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% Glycerol, 0.1% Tween, 0.2 mM 1.4-Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl fluoride (PMSF) and Roche protease inhibitor tablets). .. The cell lysate was clarified by centrifugation and precipitated with 65% ammonium sulfate.

Article Title: A novel HDAC inhibitor, CG200745, inhibits pancreatic cancer cell growth and overcomes gemcitabine resistance
Article Snippet: Values are the means of triplicate wells from three independent experiments for each drug concentration. .. Cells were lysed in a lysis buffer containing 70 mM glycerophosphate (pH 7.2), 0.6 mM Na vanadate, 2 mM MgCl2 , 1 mM EGTA, 1 mM DTT, 0.5% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 1× complete protease inhibitor (Roche Applied Science, Nutley, NJ, USA). .. Protein (25 μg) was resolved on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford, MA, USA).

Article Title: Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation
Article Snippet: mRNA coding for human DPCK domain (COASY amino acid sequence from 355 to 564) was expressed with the N-terminal histidine-tag from pET30-a(+) (Novagen) at 37°C in E. coli strain BL21 (DE3), after induction with 0.2 mM IPTG. .. Cells were lysed by a French press in 50 mM Tris-HCl (pH 8), 0.5 M NaCl, 1% Triton X-100, 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM β-mercaptoethanol, and Roche Complete EDTA-free protease inhibitor cocktail. .. After clearing, the lysate was loaded on a Ni-NTA beads (QIAGEN) column.

Article Title: Identification and characterization of the role of c-terminal Src kinase in dengue virus replication
Article Snippet: Real time PCRs were performed by using one-step or two-step methods using SYBR green or Taqman chemistry as described previously . .. For immunoblotting, cell lysates were prepared in lysis buffer (50 mM Tris-Cl pH-8.0, 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS) containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (PIC) (Roche). .. Supernatants obtained after centrifugation at 17,000× g were used for loading onto SDS-PAGE and analysed by western blotting as described previously .

Article Title: Efficacy of EZH2 inhibitory drugs in human papillomavirus-positive and human papillomavirus-negative oropharyngeal squamous cell carcinomas
Article Snippet: Contrast and sharpness of images were corrected manually for purposes of clarity utilizing Photoshop CC (Adobe Systems). .. Whole cell lysates were utilized in the analysis of EZH2 protein content and were obtained from harvesting cells in 200 μL 50 mM TRIS +1% SDS pH 7.6, supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 × complete protease inhibitor (CPI) (Cat#: 11697498001 Roche). .. Cells were incubated on ice for 20 min, sheared through a 25-gauge syringe, and centrifuged for 15 min at 16,000 ×g at 4 °C.

Article Title: Changes of Cell Biochemical States Are Revealed in Protein Homomeric Complex Dynamics
Article Snippet: The TAP-tagged LEU1-TAP BY4741 strain (Dharmacon-Horizon Discovery, Cambridge, UK) was grown in 50 mM metformin containing YPD or plain YPD broth until the culture reached an O.D.600 nm of 1.0 (4 replicates for each condition). .. The yeast cells were then harvested by centrifugation, washed and lysed by beadbeating and vortexing 25 times for one minute in lysis buffer (150 mM of NaCl, 50 mM of Tris-HCl pH 8.0, 5 mM of EDTA, 10% (v/v) glycerol, 0.2% (v/v) Nonidet P-40, 1 mM of NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete mini EDTA-free protease inhibitor cocktail from Roche, Mannheim, Germany; ). .. The lysed yeast cell supernatants for both conditions were clarified by centrifugation at 21,000 g for 30 min, and the protein concentrations were determined by a Bradford assay ( ).

Article Title: Rad51 Interacts with Non-structural 3 Protein of Hepatitis C Virus and Regulates Viral Production
Article Snippet: At 48 h after transfection, cells were harvested and immunoprecipitation assay was performed as we reported previously ( ). .. Cells were harvested and lysed in buffer containing 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.25% sodium deoxycholate, 1 mM Na3 VO4 , 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM β-glycerophosphate, and protease inhibitor cocktail (Roche) for 30 min on ice, and centrifuged at 12,000 rpm for 10 min at 4°C. .. The supernatant was collected and equal amounts of protein were subjected to SDS-PAGE and electrotransferred to a nitrocellulose membrane.

Article Title: Differential PKA activation and AKAP association determines cell fate in cancer cells
Article Snippet: The AKAP inhibitor Ht31 was obtained from Promega (# V8211). .. Cells were lysed in TNESV lysis buffer (50 mmol/liter Tris (pH 7.5), 150 mmol/liter NaCl, 1% NP40, 50 mmol/liter NaF, 1 mmol/liter Na3VO4, 25 μg/ml β-glycerophosphate, 1 mmol/liter phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Roche, Indianapolis, IN) as previously described [ , , , ]. .. Western blotting was performed using previous established protocol from our laboratory [ , , - ].

Article Title: Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens
Article Snippet: At OD600 =0.75 protein expression was induced with 1 mM isopropyl β-D -1-thiogalactopyranoside (IPTG), and the cells grown overnight at 20 °C. .. Cells were harvested by centrifugation, and cell pellets dissolved in 10 ml fusion protein lysis buffer (50 mM Tris, 25 mM NaCl, 5% glycerol, pH 7.8) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail mixture (Roche) per litre of starting culture. .. Lysozyme (Merck) was added to a final concentration of 1 mg ml−1 and the cell suspension was incubated for 1 h on ice.

Article Title: Clinically used antirheumatic agent auranofin is a proteasomal deubiquitinase inhibitor and inhibits tumor growth
Article Snippet: Lipofectamine™ RNAiMAX and Lipofectamine 2000 were purchased from Invitrogen Corporation. .. Whole cell lysates were prepared in RIPA buffer supplemented with 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1×Roche Complete Mini Protease Inhibitor Cocktail (Roche, Indianapolis, IN). .. To detect the level of IκBα in the cytosol and NF-κB p65 in the nuclear, cytosol and the nuclear fractions were extracted by using a kit from Nanjing Keygen (Nanjing, China).

Infection:

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability
Article Snippet: HN30 cells were cultured in DMEM medium supplemented with 10% FBS. .. To detect BirA*-RASG12V proteins and biotin labeling, HEK-HT cells or HEK-HT cells stably infected with LentiCRISPR2.0 or LentiCRISPR2.0-sgPIP5K1A-1, -2, or -3 were transiently transfected with pcDNA3.1-myc-BirA*-KRASG12V , -NRASG12V , and/or -HRASG12V were treated with biotin (see the Biotin identification section below) and then lysed in buffer containing 1% NP-40, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Roche) by end-to-end incubation at 4 °C for 30 min. Lysates were centrifuged at 21,000 x g for 10 min. Total protein was quantified using BCA kit (Thermo Fisher Scientific). .. Proteins were separated on 12.5% or gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad) and transferred onto polyvinylidene difluoride membranes and probed with anti-myc (Cell Signaling, #2276; 1:1000) or β-actin (Cell Signaling #3700; 1:5000) primary antibodies in blocking buffer containing either 5% milk or 5% bovine serum albumin followed by the secondary antibodies of goat anti-rabbit IgG (H+L) HRP (Thermo Fisher Scientific, #65-6120, 1:2000) or goat anti-mouse IgG (H+L) HRP (Life Technologies, #G21040, 1:5000).

Purification:

Article Title: The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition
Article Snippet: Paragraph title: Expression and purification of recombinant proteins ... The pellet was re-suspended in buffer A (50 mM Tris–HCl pH8.0, 300 mM NaCl) containing 2 mM phenylmethanesulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablets (Roche).

Article Title: Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE
Article Snippet: The untagged TWINKLE was purified using N-His6 -SUMO-fused TWINKLE construct made using Champion pET SUMO expression system from Invitrogen and Escherichia coli Rosetta (DE3) cells. .. Briefly, cells were lysed using lysozyme (0.2 mg/ml) and repeated freeze-thaw cycles in liquid nitrogen after pellet resuspension in Buffer A (50 mM Tris Cl pH 7.1, 600 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% Glycerol, 0.1% Tween, 0.2 mM 1.4-Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl fluoride (PMSF) and Roche protease inhibitor tablets).

Article Title: Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation
Article Snippet: Paragraph title: Expression and Purification of Recombinant hDPCK in Bacteria ... Cells were lysed by a French press in 50 mM Tris-HCl (pH 8), 0.5 M NaCl, 1% Triton X-100, 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM β-mercaptoethanol, and Roche Complete EDTA-free protease inhibitor cocktail.

Article Title: Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins
Article Snippet: After clearing the lysate by centrifugation for 60 min at 20’000 rpm, 4 °C, the Cglu PafA was purified by nickel affinity chromatography using a 5 ml HisTrap HP column (GE Healthcare). .. The cells were harvested and the pellets were resuspended in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, supplemented with DNAse, Phenylmethylsulfonyl fluoride (PMSF) and Inhibitor Cocktail (Roche) and subsequently lysed by passing the solution four times through an Emulsiflex High Pressure Homogenizer.

Article Title: Changes of Cell Biochemical States Are Revealed in Protein Homomeric Complex Dynamics
Article Snippet: The yeast cells were then harvested by centrifugation, washed and lysed by beadbeating and vortexing 25 times for one minute in lysis buffer (150 mM of NaCl, 50 mM of Tris-HCl pH 8.0, 5 mM of EDTA, 10% (v/v) glycerol, 0.2% (v/v) Nonidet P-40, 1 mM of NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete mini EDTA-free protease inhibitor cocktail from Roche, Mannheim, Germany; ). .. The yeast cells were then harvested by centrifugation, washed and lysed by beadbeating and vortexing 25 times for one minute in lysis buffer (150 mM of NaCl, 50 mM of Tris-HCl pH 8.0, 5 mM of EDTA, 10% (v/v) glycerol, 0.2% (v/v) Nonidet P-40, 1 mM of NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete mini EDTA-free protease inhibitor cocktail from Roche, Mannheim, Germany; ).

Article Title: Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens
Article Snippet: Paragraph title: Expression and purification of TXNDC11 constructs containing the coiled-coil domain ... Cells were harvested by centrifugation, and cell pellets dissolved in 10 ml fusion protein lysis buffer (50 mM Tris, 25 mM NaCl, 5% glycerol, pH 7.8) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail mixture (Roche) per litre of starting culture.

Imaging:

Article Title: Phagocytes from Mice Lacking the Sts Phosphatases Have an Enhanced Antifungal Response to Candida albicans
Article Snippet: Cells obtained from male or female mice were stimulated, washed, and lysed in buffer containing 0.05 M Tris, 0.15 M sodium chloride, 5 µM EDTA, 0.2 mM pervanadate, 0.5 mg/ml phenylmethylsulfonyl fluoride (PMSF), and 1× Roche protease inhibitors. .. Cells obtained from male or female mice were stimulated, washed, and lysed in buffer containing 0.05 M Tris, 0.15 M sodium chloride, 5 µM EDTA, 0.2 mM pervanadate, 0.5 mg/ml phenylmethylsulfonyl fluoride (PMSF), and 1× Roche protease inhibitors.

Protein Concentration:

Article Title: Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain
Article Snippet: Frozen tissue was homogenized (0.5–1 mg mL−1 ) in ice-cold lysis buffer containing 50 mM Tris-buffered saline (TBS, pH 7.4), 0.1 % (v/v) Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM ethylene glycol tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete™ protease inhibitor (Roche Diagnostics Ltd., West Sussex, UK). .. Protein concentrations in supernatants were measured using a BCA protein assay kit (Pierce Endogen, Rockford, USA).

Sequencing:

Article Title: Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation
Article Snippet: mRNA coding for human DPCK domain (COASY amino acid sequence from 355 to 564) was expressed with the N-terminal histidine-tag from pET30-a(+) (Novagen) at 37°C in E. coli strain BL21 (DE3), after induction with 0.2 mM IPTG. .. Cells were lysed by a French press in 50 mM Tris-HCl (pH 8), 0.5 M NaCl, 1% Triton X-100, 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM β-mercaptoethanol, and Roche Complete EDTA-free protease inhibitor cocktail.

Sonication:

Article Title: Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain
Article Snippet: Frozen tissue was homogenized (0.5–1 mg mL−1 ) in ice-cold lysis buffer containing 50 mM Tris-buffered saline (TBS, pH 7.4), 0.1 % (v/v) Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM ethylene glycol tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete™ protease inhibitor (Roche Diagnostics Ltd., West Sussex, UK). .. Samples were normalised to equal protein concentration before being analyzed by western blotting or ELISA.

Article Title: Source of the Fitness Defect in Rifamycin-Resistant Mycobacterium tuberculosis RNA Polymerase and the Mechanism of Compensation by Mutations in the β′ Subunit
Article Snippet: Protein expression was induced by addition of 1 mM IPTG (isopropyl-β- d -thiogalactopyranoside). .. Cells were lysed via sonication in 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol (β-ME), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1× Roche cOmplete ULTRA protease cocktail, 200 U of DNase I, and 1 mg/ml lysozyme. .. Clarified lysate was passed through a 0.22-μm-pore size filter and applied to a HisTrap HP column (GE Healthcare).

Binding Assay:

Article Title: Differential PKA activation and AKAP association determines cell fate in cancer cells
Article Snippet: Cells were lysed in TNESV lysis buffer (50 mmol/liter Tris (pH 7.5), 150 mmol/liter NaCl, 1% NP40, 50 mmol/liter NaF, 1 mmol/liter Na3VO4, 25 μg/ml β-glycerophosphate, 1 mmol/liter phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Roche, Indianapolis, IN) as previously described [ , , , ]. .. Co- Immunoprecipitation (IP) was performed with 500 μg protein aliquots using magnetic beads (Millipore) according to the instructions provided by the manufacturer (# LSKMAGAG10) and previously established method [ ].

Article Title: Acidified bile acids enhance tumor progression and telomerase activity of gastric cancer in mice dependent on c‐Myc expression
Article Snippet: All protein samples were homogenized on ice in lysis buffer containing 40 mmol/L Tris‐HCl (pH 6.9), 2 mmol/L ethylenediaminetetraacetic acid (EDTA, pH 8.0), 100 mmol/L sodium fluoride, 150 mmol/L NaCl, 10 mmol/L sodium pyrophosphate, 1% Tergitol type NP‐40, 2 mmol/L sodium orthovanadate, 1% Triton X‐100, 1.0 mmol/L phenylmethanesulfonyl fluoride (PMSF), and 1 × protein inhibitor mini‐tablet (Roche, Basel, Switzerland). .. Next, equal amounts (30 μ g) of protein were heated at 95°C for 5 min in 5 × Laemmli sample buffer and then separated on a 10% SDS‐PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes by wet transfer method.

Molecular Weight:

Article Title: Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins
Article Snippet: The cells were harvested and the pellets were resuspended in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, supplemented with DNAse, Phenylmethylsulfonyl fluoride (PMSF) and Inhibitor Cocktail (Roche) and subsequently lysed by passing the solution four times through an Emulsiflex High Pressure Homogenizer. .. The solution was cleared by centrifugation at 20’000 rpm for 60 min at 4 °C and the protein was purified by nickel affinity chromatography using a 5 ml HisTrap HP column (GE Healthcare).

Real-time Polymerase Chain Reaction:

Article Title: Identification and characterization of the role of c-terminal Src kinase in dengue virus replication
Article Snippet: Paragraph title: Viral titers, real time PCR and western blotting ... For immunoblotting, cell lysates were prepared in lysis buffer (50 mM Tris-Cl pH-8.0, 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS) containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (PIC) (Roche).

Magnetic Beads:

Article Title: Differential PKA activation and AKAP association determines cell fate in cancer cells
Article Snippet: Cells were lysed in TNESV lysis buffer (50 mmol/liter Tris (pH 7.5), 150 mmol/liter NaCl, 1% NP40, 50 mmol/liter NaF, 1 mmol/liter Na3VO4, 25 μg/ml β-glycerophosphate, 1 mmol/liter phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Roche, Indianapolis, IN) as previously described [ , , , ]. .. Cells were lysed in TNESV lysis buffer (50 mmol/liter Tris (pH 7.5), 150 mmol/liter NaCl, 1% NP40, 50 mmol/liter NaF, 1 mmol/liter Na3VO4, 25 μg/ml β-glycerophosphate, 1 mmol/liter phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Roche, Indianapolis, IN) as previously described [ , , , ].

Isolation:

Article Title: Identification and characterization of the role of c-terminal Src kinase in dengue virus replication
Article Snippet: For quantitation of RNA, cells were washed once with PBS and collected in 250 μl of Trizol reagent (Life Technologies) for isolation of total RNA. .. For immunoblotting, cell lysates were prepared in lysis buffer (50 mM Tris-Cl pH-8.0, 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS) containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (PIC) (Roche).

Transfection:

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability
Article Snippet: HN30 cells were cultured in DMEM medium supplemented with 10% FBS. .. To detect BirA*-RASG12V proteins and biotin labeling, HEK-HT cells or HEK-HT cells stably infected with LentiCRISPR2.0 or LentiCRISPR2.0-sgPIP5K1A-1, -2, or -3 were transiently transfected with pcDNA3.1-myc-BirA*-KRASG12V , -NRASG12V , and/or -HRASG12V were treated with biotin (see the Biotin identification section below) and then lysed in buffer containing 1% NP-40, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Roche) by end-to-end incubation at 4 °C for 30 min. Lysates were centrifuged at 21,000 x g for 10 min. Total protein was quantified using BCA kit (Thermo Fisher Scientific). .. Proteins were separated on 12.5% or gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad) and transferred onto polyvinylidene difluoride membranes and probed with anti-myc (Cell Signaling, #2276; 1:1000) or β-actin (Cell Signaling #3700; 1:5000) primary antibodies in blocking buffer containing either 5% milk or 5% bovine serum albumin followed by the secondary antibodies of goat anti-rabbit IgG (H+L) HRP (Thermo Fisher Scientific, #65-6120, 1:2000) or goat anti-mouse IgG (H+L) HRP (Life Technologies, #G21040, 1:5000).

Labeling:

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability
Article Snippet: HN30 cells were cultured in DMEM medium supplemented with 10% FBS. .. To detect BirA*-RASG12V proteins and biotin labeling, HEK-HT cells or HEK-HT cells stably infected with LentiCRISPR2.0 or LentiCRISPR2.0-sgPIP5K1A-1, -2, or -3 were transiently transfected with pcDNA3.1-myc-BirA*-KRASG12V , -NRASG12V , and/or -HRASG12V were treated with biotin (see the Biotin identification section below) and then lysed in buffer containing 1% NP-40, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Roche) by end-to-end incubation at 4 °C for 30 min. Lysates were centrifuged at 21,000 x g for 10 min. Total protein was quantified using BCA kit (Thermo Fisher Scientific). .. Proteins were separated on 12.5% or gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad) and transferred onto polyvinylidene difluoride membranes and probed with anti-myc (Cell Signaling, #2276; 1:1000) or β-actin (Cell Signaling #3700; 1:5000) primary antibodies in blocking buffer containing either 5% milk or 5% bovine serum albumin followed by the secondary antibodies of goat anti-rabbit IgG (H+L) HRP (Thermo Fisher Scientific, #65-6120, 1:2000) or goat anti-mouse IgG (H+L) HRP (Life Technologies, #G21040, 1:5000).

Article Title: Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins
Article Snippet: The cells were harvested and the pellets were resuspended in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, supplemented with DNAse, Phenylmethylsulfonyl fluoride (PMSF) and Inhibitor Cocktail (Roche) and subsequently lysed by passing the solution four times through an Emulsiflex High Pressure Homogenizer. .. To remove uncleaved protein, the cleaved hexahistidine tag and the TEV protease, the protein solution was again passed over the 5 ml HisTrap HP column and further purified using a Superose 6 pg in buffer containing 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, 1 mM DTT, 1 mM EDTA and 10% glycerol.

Size-exclusion Chromatography:

Article Title: The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition
Article Snippet: The pellet was re-suspended in buffer A (50 mM Tris–HCl pH8.0, 300 mM NaCl) containing 2 mM phenylmethanesulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablets (Roche). .. The supernatant was incubated with Ni-NTA resin (Sigma–Aldrich) for 1 h. The protein was then eluted using buffer A with a gradient of imidazole (10–200 mM).

Article Title: Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation
Article Snippet: Cells were lysed by a French press in 50 mM Tris-HCl (pH 8), 0.5 M NaCl, 1% Triton X-100, 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM β-mercaptoethanol, and Roche Complete EDTA-free protease inhibitor cocktail. .. Cells were lysed by a French press in 50 mM Tris-HCl (pH 8), 0.5 M NaCl, 1% Triton X-100, 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM β-mercaptoethanol, and Roche Complete EDTA-free protease inhibitor cocktail.

Article Title: Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins
Article Snippet: Size exclusion chromatography was carried out on a Superose 6 pg gel filtration column equilibrated and run in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, 1 mM DTT, 1 mM EDTA and 10% glycerol. .. The cells were harvested and the pellets were resuspended in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, supplemented with DNAse, Phenylmethylsulfonyl fluoride (PMSF) and Inhibitor Cocktail (Roche) and subsequently lysed by passing the solution four times through an Emulsiflex High Pressure Homogenizer.

Protein Purification:

Article Title: Source of the Fitness Defect in Rifamycin-Resistant Mycobacterium tuberculosis RNA Polymerase and the Mechanism of Compensation by Mutations in the β′ Subunit
Article Snippet: Paragraph title: Protein purification. ... Cells were lysed via sonication in 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol (β-ME), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1× Roche cOmplete ULTRA protease cocktail, 200 U of DNase I, and 1 mg/ml lysozyme.

Affinity Chromatography:

Article Title: Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins
Article Snippet: After clearing the lysate by centrifugation for 60 min at 20’000 rpm, 4 °C, the Cglu PafA was purified by nickel affinity chromatography using a 5 ml HisTrap HP column (GE Healthcare). .. The cells were harvested and the pellets were resuspended in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, supplemented with DNAse, Phenylmethylsulfonyl fluoride (PMSF) and Inhibitor Cocktail (Roche) and subsequently lysed by passing the solution four times through an Emulsiflex High Pressure Homogenizer.

Mouse Assay:

Article Title: Phagocytes from Mice Lacking the Sts Phosphatases Have an Enhanced Antifungal Response to Candida albicans
Article Snippet: Fungal CFU were obtained by plating serial dilutions onto YPD plates. .. Cells obtained from male or female mice were stimulated, washed, and lysed in buffer containing 0.05 M Tris, 0.15 M sodium chloride, 5 µM EDTA, 0.2 mM pervanadate, 0.5 mg/ml phenylmethylsulfonyl fluoride (PMSF), and 1× Roche protease inhibitors. .. Lysates were clarified by centrifugation, subjected to SDS-PAGE, and transferred to nitrocellulose (Whatman).

Article Title: Acidified bile acids enhance tumor progression and telomerase activity of gastric cancer in mice dependent on c‐Myc expression
Article Snippet: All gastric cancer biopsies were taken from a −80°C tissue bank and animal tumor tissues were from subcutaneous transplanted xenografts in nude mice. .. All protein samples were homogenized on ice in lysis buffer containing 40 mmol/L Tris‐HCl (pH 6.9), 2 mmol/L ethylenediaminetetraacetic acid (EDTA, pH 8.0), 100 mmol/L sodium fluoride, 150 mmol/L NaCl, 10 mmol/L sodium pyrophosphate, 1% Tergitol type NP‐40, 2 mmol/L sodium orthovanadate, 1% Triton X‐100, 1.0 mmol/L phenylmethanesulfonyl fluoride (PMSF), and 1 × protein inhibitor mini‐tablet (Roche, Basel, Switzerland).

SDS Page:

Article Title: Cyclocarya paliurus tea leaves enhances pancreatic β cell preservation through inhibition of apoptosis
Article Snippet: Immunoblotting was performed as previously described . .. Briefly, proteins were extracted from the pancreas tissues and NIT-1 cells in the presence of a cocktail of protease inhibitors and phenylmethylsulfonyl fluoride (PMSF) (Roche Applied Science, Branford, CT, USA), resolved and separated by 12–15% SDS-PAGE, and transferred to PVDF membranes. .. The membranes were then probed with the primary antibodies caspase-8, caspase-9, cleaved caspase-3, Bax, Bcl-2, ERK, JNK, p38, p-ERK, p-JNK,p-p38, Akt, p-Akt and β-actin (Cell Signaling Technology, Inc. Beverly, MA, USA), followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies.

Article Title: Phagocytes from Mice Lacking the Sts Phosphatases Have an Enhanced Antifungal Response to Candida albicans
Article Snippet: Cells obtained from male or female mice were stimulated, washed, and lysed in buffer containing 0.05 M Tris, 0.15 M sodium chloride, 5 µM EDTA, 0.2 mM pervanadate, 0.5 mg/ml phenylmethylsulfonyl fluoride (PMSF), and 1× Roche protease inhibitors. .. Cells obtained from male or female mice were stimulated, washed, and lysed in buffer containing 0.05 M Tris, 0.15 M sodium chloride, 5 µM EDTA, 0.2 mM pervanadate, 0.5 mg/ml phenylmethylsulfonyl fluoride (PMSF), and 1× Roche protease inhibitors.

Article Title: Efficacy of EZH2 inhibitory drugs in human papillomavirus-positive and human papillomavirus-negative oropharyngeal squamous cell carcinomas
Article Snippet: Whole cell lysates were utilized in the analysis of EZH2 protein content and were obtained from harvesting cells in 200 μL 50 mM TRIS +1% SDS pH 7.6, supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 × complete protease inhibitor (CPI) (Cat#: 11697498001 Roche). .. Cells were incubated on ice for 20 min, sheared through a 25-gauge syringe, and centrifuged for 15 min at 16,000 ×g at 4 °C.

Article Title: Acidified bile acids enhance tumor progression and telomerase activity of gastric cancer in mice dependent on c‐Myc expression
Article Snippet: All protein samples were homogenized on ice in lysis buffer containing 40 mmol/L Tris‐HCl (pH 6.9), 2 mmol/L ethylenediaminetetraacetic acid (EDTA, pH 8.0), 100 mmol/L sodium fluoride, 150 mmol/L NaCl, 10 mmol/L sodium pyrophosphate, 1% Tergitol type NP‐40, 2 mmol/L sodium orthovanadate, 1% Triton X‐100, 1.0 mmol/L phenylmethanesulfonyl fluoride (PMSF), and 1 × protein inhibitor mini‐tablet (Roche, Basel, Switzerland). .. Protein concentration was estimated using the Pierce Protein Estimation System (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol.

Plasmid Preparation:

Article Title: The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition
Article Snippet: In each case, the plasmid with the gene of interest was transformed into Escherichia coli BL21(DE3) strain (Novagen Inc .). .. The pellet was re-suspended in buffer A (50 mM Tris–HCl pH8.0, 300 mM NaCl) containing 2 mM phenylmethanesulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablets (Roche).

Article Title: Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins
Article Snippet: Briefly, Cglu PafA was expressed from an IPTG-inducible pET24(+) plasmid in E. coli Rosetta cells for 16 h at 23 °C. .. The cells were harvested and the pellets were resuspended in 50 mM Tris, pH 7.5 (4 °C), 150 mM NaCl, supplemented with DNAse, Phenylmethylsulfonyl fluoride (PMSF) and Inhibitor Cocktail (Roche) and subsequently lysed by passing the solution four times through an Emulsiflex High Pressure Homogenizer.

Enzyme-linked Immunosorbent Assay:

Article Title: Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain
Article Snippet: Frozen tissue was homogenized (0.5–1 mg mL−1 ) in ice-cold lysis buffer containing 50 mM Tris-buffered saline (TBS, pH 7.4), 0.1 % (v/v) Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM ethylene glycol tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete™ protease inhibitor (Roche Diagnostics Ltd., West Sussex, UK). .. Protein concentrations in supernatants were measured using a BCA protein assay kit (Pierce Endogen, Rockford, USA).

Recombinant:

Article Title: The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition
Article Snippet: Paragraph title: Expression and purification of recombinant proteins ... The pellet was re-suspended in buffer A (50 mM Tris–HCl pH8.0, 300 mM NaCl) containing 2 mM phenylmethanesulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablets (Roche).

Article Title: Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation
Article Snippet: Paragraph title: Expression and Purification of Recombinant hDPCK in Bacteria ... Cells were lysed by a French press in 50 mM Tris-HCl (pH 8), 0.5 M NaCl, 1% Triton X-100, 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM β-mercaptoethanol, and Roche Complete EDTA-free protease inhibitor cocktail.

Titration:

Article Title: Differential PKA activation and AKAP association determines cell fate in cancer cells
Article Snippet: Cells were lysed in TNESV lysis buffer (50 mmol/liter Tris (pH 7.5), 150 mmol/liter NaCl, 1% NP40, 50 mmol/liter NaF, 1 mmol/liter Na3VO4, 25 μg/ml β-glycerophosphate, 1 mmol/liter phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Roche, Indianapolis, IN) as previously described [ , , , ]. .. Co- Immunoprecipitation (IP) was performed with 500 μg protein aliquots using magnetic beads (Millipore) according to the instructions provided by the manufacturer (# LSKMAGAG10) and previously established method [ ].

Quantitation Assay:

Article Title: Identification and characterization of the role of c-terminal Src kinase in dengue virus replication
Article Snippet: For quantitation of RNA, cells were washed once with PBS and collected in 250 μl of Trizol reagent (Life Technologies) for isolation of total RNA. .. For immunoblotting, cell lysates were prepared in lysis buffer (50 mM Tris-Cl pH-8.0, 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS) containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (PIC) (Roche).

Immunoprecipitation:

Article Title: Differential PKA activation and AKAP association determines cell fate in cancer cells
Article Snippet: Paragraph title: Antibodies, western blotting and immunoprecipitation ... Cells were lysed in TNESV lysis buffer (50 mmol/liter Tris (pH 7.5), 150 mmol/liter NaCl, 1% NP40, 50 mmol/liter NaF, 1 mmol/liter Na3VO4, 25 μg/ml β-glycerophosphate, 1 mmol/liter phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Roche, Indianapolis, IN) as previously described [ , , , ].

Lysis:

Article Title: Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain
Article Snippet: Frozen postmortem human temporal cortex (Table 1) from control (n = 5) and pathologically confirmed cases of sporadic AD of Braak stage II (n = 5), III (3), IV (n = 4), V (n = 3) and VI (n = 5) were obtained from the MRC London Neurodegenerative Diseases Brain Bank (Additional file : Table S1, Additional file : Table S2). .. Frozen tissue was homogenized (0.5–1 mg mL−1 ) in ice-cold lysis buffer containing 50 mM Tris-buffered saline (TBS, pH 7.4), 0.1 % (v/v) Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM ethylene glycol tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete™ protease inhibitor (Roche Diagnostics Ltd., West Sussex, UK). .. Homogenates were centrifuged at 25,000g(av) for 20 min at 4 °C.

Article Title: Phagocytes from Mice Lacking the Sts Phosphatases Have an Enhanced Antifungal Response to Candida albicans
Article Snippet: Cells obtained from male or female mice were stimulated, washed, and lysed in buffer containing 0.05 M Tris, 0.15 M sodium chloride, 5 µM EDTA, 0.2 mM pervanadate, 0.5 mg/ml phenylmethylsulfonyl fluoride (PMSF), and 1× Roche protease inhibitors. .. Cells obtained from male or female mice were stimulated, washed, and lysed in buffer containing 0.05 M Tris, 0.15 M sodium chloride, 5 µM EDTA, 0.2 mM pervanadate, 0.5 mg/ml phenylmethylsulfonyl fluoride (PMSF), and 1× Roche protease inhibitors.

Article Title: A novel HDAC inhibitor, CG200745, inhibits pancreatic cancer cell growth and overcomes gemcitabine resistance
Article Snippet: Values are the means of triplicate wells from three independent experiments for each drug concentration. .. Cells were lysed in a lysis buffer containing 70 mM glycerophosphate (pH 7.2), 0.6 mM Na vanadate, 2 mM MgCl2 , 1 mM EGTA, 1 mM DTT, 0.5% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 1× complete protease inhibitor (Roche Applied Science, Nutley, NJ, USA). .. Protein (25 μg) was resolved on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford, MA, USA).

Article Title: Identification and characterization of the role of c-terminal Src kinase in dengue virus replication
Article Snippet: Real time PCRs were performed by using one-step or two-step methods using SYBR green or Taqman chemistry as described previously . .. For immunoblotting, cell lysates were prepared in lysis buffer (50 mM Tris-Cl pH-8.0, 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS) containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (PIC) (Roche). .. Supernatants obtained after centrifugation at 17,000× g were used for loading onto SDS-PAGE and analysed by western blotting as described previously .

Article Title: Changes of Cell Biochemical States Are Revealed in Protein Homomeric Complex Dynamics
Article Snippet: The TAP-tagged LEU1-TAP BY4741 strain (Dharmacon-Horizon Discovery, Cambridge, UK) was grown in 50 mM metformin containing YPD or plain YPD broth until the culture reached an O.D.600 nm of 1.0 (4 replicates for each condition). .. The yeast cells were then harvested by centrifugation, washed and lysed by beadbeating and vortexing 25 times for one minute in lysis buffer (150 mM of NaCl, 50 mM of Tris-HCl pH 8.0, 5 mM of EDTA, 10% (v/v) glycerol, 0.2% (v/v) Nonidet P-40, 1 mM of NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete mini EDTA-free protease inhibitor cocktail from Roche, Mannheim, Germany; ). .. The lysed yeast cell supernatants for both conditions were clarified by centrifugation at 21,000 g for 30 min, and the protein concentrations were determined by a Bradford assay ( ).

Article Title: Differential PKA activation and AKAP association determines cell fate in cancer cells
Article Snippet: The AKAP inhibitor Ht31 was obtained from Promega (# V8211). .. Cells were lysed in TNESV lysis buffer (50 mmol/liter Tris (pH 7.5), 150 mmol/liter NaCl, 1% NP40, 50 mmol/liter NaF, 1 mmol/liter Na3VO4, 25 μg/ml β-glycerophosphate, 1 mmol/liter phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Roche, Indianapolis, IN) as previously described [ , , , ]. .. Western blotting was performed using previous established protocol from our laboratory [ , , - ].

Article Title: Acidified bile acids enhance tumor progression and telomerase activity of gastric cancer in mice dependent on c‐Myc expression
Article Snippet: All gastric cancer biopsies were taken from a −80°C tissue bank and animal tumor tissues were from subcutaneous transplanted xenografts in nude mice. .. All protein samples were homogenized on ice in lysis buffer containing 40 mmol/L Tris‐HCl (pH 6.9), 2 mmol/L ethylenediaminetetraacetic acid (EDTA, pH 8.0), 100 mmol/L sodium fluoride, 150 mmol/L NaCl, 10 mmol/L sodium pyrophosphate, 1% Tergitol type NP‐40, 2 mmol/L sodium orthovanadate, 1% Triton X‐100, 1.0 mmol/L phenylmethanesulfonyl fluoride (PMSF), and 1 × protein inhibitor mini‐tablet (Roche, Basel, Switzerland). .. Protein concentration was estimated using the Pierce Protein Estimation System (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol.

Article Title: Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens
Article Snippet: At OD600 =0.75 protein expression was induced with 1 mM isopropyl β-D -1-thiogalactopyranoside (IPTG), and the cells grown overnight at 20 °C. .. Cells were harvested by centrifugation, and cell pellets dissolved in 10 ml fusion protein lysis buffer (50 mM Tris, 25 mM NaCl, 5% glycerol, pH 7.8) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail mixture (Roche) per litre of starting culture. .. Lysozyme (Merck) was added to a final concentration of 1 mg ml−1 and the cell suspension was incubated for 1 h on ice.

Positron Emission Tomography:

Article Title: Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE
Article Snippet: The untagged TWINKLE was purified using N-His6 -SUMO-fused TWINKLE construct made using Champion pET SUMO expression system from Invitrogen and Escherichia coli Rosetta (DE3) cells. .. Briefly, cells were lysed using lysozyme (0.2 mg/ml) and repeated freeze-thaw cycles in liquid nitrogen after pellet resuspension in Buffer A (50 mM Tris Cl pH 7.1, 600 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% Glycerol, 0.1% Tween, 0.2 mM 1.4-Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl fluoride (PMSF) and Roche protease inhibitor tablets).

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    Roche phenylmethylsulfonyl fluoride pmsf
    Phenylmethylsulfonyl Fluoride Pmsf, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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