Structured Review

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Cells in Figure 5 entered the MI-MII transition at the time of kinase inhibition. ( A – D ) Cell–cycle stage quantification for Figure 5A–5D , respectively. At the time of treatment with 10 µM 1–NM–PP1 and 20 µM 1–NA–PP1, all strains had ~50% of cells in Anaphase I. The 8 hr 30’ immunofluorescence time point is from cells in this same experiment that did not receive the kinase-inhibitor treatment, 45 min after the other cells did receive the inhibitor treatment. The purpose of this time point is to demonstrate that all four strains would successfully <t>complete</t> meiosis I had they not been received any kinase inhibitor.
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Images

1) Product Images from "Multiple kinases inhibit origin licensing and helicase activation to ensure reductive cell division during meiosis"

Article Title: Multiple kinases inhibit origin licensing and helicase activation to ensure reductive cell division during meiosis

Journal: eLife

doi: 10.7554/eLife.33309

Cells in Figure 5 entered the MI-MII transition at the time of kinase inhibition. ( A – D ) Cell–cycle stage quantification for Figure 5A–5D , respectively. At the time of treatment with 10 µM 1–NM–PP1 and 20 µM 1–NA–PP1, all strains had ~50% of cells in Anaphase I. The 8 hr 30’ immunofluorescence time point is from cells in this same experiment that did not receive the kinase-inhibitor treatment, 45 min after the other cells did receive the inhibitor treatment. The purpose of this time point is to demonstrate that all four strains would successfully complete meiosis I had they not been received any kinase inhibitor.
Figure Legend Snippet: Cells in Figure 5 entered the MI-MII transition at the time of kinase inhibition. ( A – D ) Cell–cycle stage quantification for Figure 5A–5D , respectively. At the time of treatment with 10 µM 1–NM–PP1 and 20 µM 1–NA–PP1, all strains had ~50% of cells in Anaphase I. The 8 hr 30’ immunofluorescence time point is from cells in this same experiment that did not receive the kinase-inhibitor treatment, 45 min after the other cells did receive the inhibitor treatment. The purpose of this time point is to demonstrate that all four strains would successfully complete meiosis I had they not been received any kinase inhibitor.

Techniques Used: Inhibition, Immunofluorescence

2) Product Images from "Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism"

Article Title: Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1817759116

Compatibility of interaction interfaces in MICU2 and MICU1 homodimers. ( A ) The dimer of MICU2 (6EAZ) is shown on the left, a dimer of MICU1 (4NSC, chains C and E) is shown in the middle, and the superimposition of the MICU1 and MICU2 dimers is shown on the right (only the core domains are shown). Red boxes indicate the EF1–EF3 interaction region, which is shown in more detail in B – D . Note that due to the symmetry the complete interaction interface consists of two such regions. ( B ) MICU2 interaction interface. Residues for the C-lobe EF3 (chain B) interaction with the N-lobe EF1 (chain A) are shown. Side chains of residues that make significant contribution to the interaction are shown in stick representation. Residues in positions −6, −5, −3, −2, +3, and +7 with respect to the loop and the L2 position within the loop are shown for EF1. Residues in positions −9, −6, −5, −2, +3, and +7 with respect to the loop and the L2 and L3 position within the loop are shown for EF3. These residues are both conserved in the MICU family, in contrast to the typical EF-hand signature, and show significant interaction ( D – F ). Residues −2 and +7 with respect to the loop are labeled. ( C ) MICU1 interface residues for the C-lobe EF3 (E chain) interaction with the N-lobe EF1 (C chain) are shown (from 4NSC). Residues in similar positions as in B are shown as sticks and/or emphasized with labeling. ( D ) The putative MICU1–MICU2 interface is shown from superimposition of the MICU1 and MICU2 dimers. The same residues as in B and C are shown as sticks and/or emphasized with labeling. ( E – G ) Protein sequence logos are shown for ( E ) MICU EF-hand-1, ( F ) MICU EF-hand-3, and ( G ) from alignments of ∼500 metazoan MICU1/2/3 sequences. The average BSA for each residue, as calculated by the PISA server, is shown above the sequence logo for MICU EF-hand 1 ( E ) and EF-hand 3 ( F ), with the orange bar indicating the BSA for the residue in MICU1 (average of 6) and the green bar for the residue in MICU2 (average of 2). Residues that are conserved in the MICU family but not in the PROSITE EF-hand motif and make significant contribution to the interaction are emphasized on the MICU logos in black.
Figure Legend Snippet: Compatibility of interaction interfaces in MICU2 and MICU1 homodimers. ( A ) The dimer of MICU2 (6EAZ) is shown on the left, a dimer of MICU1 (4NSC, chains C and E) is shown in the middle, and the superimposition of the MICU1 and MICU2 dimers is shown on the right (only the core domains are shown). Red boxes indicate the EF1–EF3 interaction region, which is shown in more detail in B – D . Note that due to the symmetry the complete interaction interface consists of two such regions. ( B ) MICU2 interaction interface. Residues for the C-lobe EF3 (chain B) interaction with the N-lobe EF1 (chain A) are shown. Side chains of residues that make significant contribution to the interaction are shown in stick representation. Residues in positions −6, −5, −3, −2, +3, and +7 with respect to the loop and the L2 position within the loop are shown for EF1. Residues in positions −9, −6, −5, −2, +3, and +7 with respect to the loop and the L2 and L3 position within the loop are shown for EF3. These residues are both conserved in the MICU family, in contrast to the typical EF-hand signature, and show significant interaction ( D – F ). Residues −2 and +7 with respect to the loop are labeled. ( C ) MICU1 interface residues for the C-lobe EF3 (E chain) interaction with the N-lobe EF1 (C chain) are shown (from 4NSC). Residues in similar positions as in B are shown as sticks and/or emphasized with labeling. ( D ) The putative MICU1–MICU2 interface is shown from superimposition of the MICU1 and MICU2 dimers. The same residues as in B and C are shown as sticks and/or emphasized with labeling. ( E – G ) Protein sequence logos are shown for ( E ) MICU EF-hand-1, ( F ) MICU EF-hand-3, and ( G ) from alignments of ∼500 metazoan MICU1/2/3 sequences. The average BSA for each residue, as calculated by the PISA server, is shown above the sequence logo for MICU EF-hand 1 ( E ) and EF-hand 3 ( F ), with the orange bar indicating the BSA for the residue in MICU1 (average of 6) and the green bar for the residue in MICU2 (average of 2). Residues that are conserved in the MICU family but not in the PROSITE EF-hand motif and make significant contribution to the interaction are emphasized on the MICU logos in black.

Techniques Used: Labeling, Sequencing

3) Product Images from "The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome"

Article Title: The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040440

TRIM50-related aggresome and TRIM50 itself are partially degraded through the autophagy-lysosomal pathway. (A) FLAG-TRIM50#3 cells were treated with 20 mM NH 4 Cl (A) and DMSO for a period of 10 hours. Cells were lysed at the indicated time after the initiation of treatment and analysed by Western blotted using FLAG and GAPDH antibodies, respectively. The experiments were performed three times and typical results are shown. (B) Curves describing TRIM50 levels as a function of time, based on the results in panel A. The density of each band was determined by densitometer. The half-time of TRIM50 was determined by calculating the protein level at each time, normalized to the corresponding GAPDH level, to the initial amount of TRIM50 protein and compared with the control cells treated with DMSO. (C) HeLa cells overexpressing EGFP-TRIM50 were incubated in complete medium (a–c), EBSS (d–f), and DMEM supplemented with NH 4 Cl 20 mM for 2 h respectively and stained with an anti-FK2 antibody.
Figure Legend Snippet: TRIM50-related aggresome and TRIM50 itself are partially degraded through the autophagy-lysosomal pathway. (A) FLAG-TRIM50#3 cells were treated with 20 mM NH 4 Cl (A) and DMSO for a period of 10 hours. Cells were lysed at the indicated time after the initiation of treatment and analysed by Western blotted using FLAG and GAPDH antibodies, respectively. The experiments were performed three times and typical results are shown. (B) Curves describing TRIM50 levels as a function of time, based on the results in panel A. The density of each band was determined by densitometer. The half-time of TRIM50 was determined by calculating the protein level at each time, normalized to the corresponding GAPDH level, to the initial amount of TRIM50 protein and compared with the control cells treated with DMSO. (C) HeLa cells overexpressing EGFP-TRIM50 were incubated in complete medium (a–c), EBSS (d–f), and DMEM supplemented with NH 4 Cl 20 mM for 2 h respectively and stained with an anti-FK2 antibody.

Techniques Used: Western Blot, Incubation, Staining

4) Product Images from "Multiple kinases inhibit origin licensing and helicase activation to ensure reductive cell division during meiosis"

Article Title: Multiple kinases inhibit origin licensing and helicase activation to ensure reductive cell division during meiosis

Journal: eLife

doi: 10.7554/eLife.33309

Cells in Figure 5 entered the MI-MII transition at the time of kinase inhibition. ( A – D ) Cell–cycle stage quantification for Figure 5A–5D , respectively. At the time of treatment with 10 µM 1–NM–PP1 and 20 µM 1–NA–PP1, all strains had ~50% of cells in Anaphase I. The 8 hr 30’ immunofluorescence time point is from cells in this same experiment that did not receive the kinase-inhibitor treatment, 45 min after the other cells did receive the inhibitor treatment. The purpose of this time point is to demonstrate that all four strains would successfully complete meiosis I had they not been received any kinase inhibitor.
Figure Legend Snippet: Cells in Figure 5 entered the MI-MII transition at the time of kinase inhibition. ( A – D ) Cell–cycle stage quantification for Figure 5A–5D , respectively. At the time of treatment with 10 µM 1–NM–PP1 and 20 µM 1–NA–PP1, all strains had ~50% of cells in Anaphase I. The 8 hr 30’ immunofluorescence time point is from cells in this same experiment that did not receive the kinase-inhibitor treatment, 45 min after the other cells did receive the inhibitor treatment. The purpose of this time point is to demonstrate that all four strains would successfully complete meiosis I had they not been received any kinase inhibitor.

Techniques Used: Inhibition, Immunofluorescence

5) Product Images from "GNB2L1 and its O-GlcNAcylation regulates metastasis via modulating epithelial-mesenchymal transition in the chemoresistance of gastric cancer"

Article Title: GNB2L1 and its O-GlcNAcylation regulates metastasis via modulating epithelial-mesenchymal transition in the chemoresistance of gastric cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0182696

OGT elevated O-GlcNAcylation on GNB2L1 in chemoresistant gastric cancer. (A-B) The O-GlcNAcylation levels of GNB2L1 in different chemoresistant cells (A) and different tissue samples (B) were assessed via IP analysis, and protein levels of OGT and OGA were also determined via WB. Progressive disease (PD) was considered as chemoresistant; complete remission (CR), partial remission (PR) and stable disease (SD) as chemosensitive. GAPDH was used as loading control.(C-E) Correlation analysis of GNB2L1 levels with OGT (D) or OGA (E) in chemoresistant gastric cancer patients. Representative images (C) and statistical data (D-E) were shown. *, P
Figure Legend Snippet: OGT elevated O-GlcNAcylation on GNB2L1 in chemoresistant gastric cancer. (A-B) The O-GlcNAcylation levels of GNB2L1 in different chemoresistant cells (A) and different tissue samples (B) were assessed via IP analysis, and protein levels of OGT and OGA were also determined via WB. Progressive disease (PD) was considered as chemoresistant; complete remission (CR), partial remission (PR) and stable disease (SD) as chemosensitive. GAPDH was used as loading control.(C-E) Correlation analysis of GNB2L1 levels with OGT (D) or OGA (E) in chemoresistant gastric cancer patients. Representative images (C) and statistical data (D-E) were shown. *, P

Techniques Used: Western Blot

Chemoresistant gastric cancer is associated with decreased protein level of GNB2L1. (A-C) Protein levels of GNB2L1 in tumor tissues from chemosensitive patients and chemoresistant patients were determined by WB (A) and IHC (B-C). Representative images (A-B) and statistical data (C) were shown. (D) Transcriptional levels of GNB2L1 in tumor tissues were determined by qPCR. (E-F) Protein levels and mRNA levels of GNB2L1 were further assessed in SGC-7901 cells and several chemoresistant cells derived from SGC-7901 cells. Progressive disease (PD) was considered as chemoresistant; complete remission (CR), partial remission (PR) and stable disease (SD) as chemosensitive. GAPDH was used as loading control. N.S., not significant; ***, P
Figure Legend Snippet: Chemoresistant gastric cancer is associated with decreased protein level of GNB2L1. (A-C) Protein levels of GNB2L1 in tumor tissues from chemosensitive patients and chemoresistant patients were determined by WB (A) and IHC (B-C). Representative images (A-B) and statistical data (C) were shown. (D) Transcriptional levels of GNB2L1 in tumor tissues were determined by qPCR. (E-F) Protein levels and mRNA levels of GNB2L1 were further assessed in SGC-7901 cells and several chemoresistant cells derived from SGC-7901 cells. Progressive disease (PD) was considered as chemoresistant; complete remission (CR), partial remission (PR) and stable disease (SD) as chemosensitive. GAPDH was used as loading control. N.S., not significant; ***, P

Techniques Used: Western Blot, Immunohistochemistry, Real-time Polymerase Chain Reaction, Derivative Assay

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Mass Spectrometry:

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BIA-KA:

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Construct:

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Incubation:

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Expressing:

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Article Snippet: The resulting C-terminal His-tagged truncated CypD was expressed in BL21(DE3) Escherichia coli (Agilent Technologies, Santa Clara, CA) by inducing expression with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 2 hours. .. Cultures were harvested by centrifugation and then re-suspended in 50 mM phosphate buffer pH 7.0, 0.5 M NaCl, 1 mM dithiothreitol (DTT) and cOmplete™ protease inhibitor (Roche Diagnostics, Mannheim, Germany).

Article Title: PIP2 stabilises active states of GPCRs and enhances the selectivity of G-protein coupling
Article Snippet: The cells from 1L expression culture were resuspended and lysed in lysis buffer (10 mM HEPES pH 7, 20 mM KCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, and cOmplete protease inhibitor (Roche)). .. The membranes were pelleted by ultracentrifugation at 108,000 × g for 35 min and solubilized in solubilisation buffer (50 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, 1% decyl-β-D-maltopyranoside (DM) (w/v), 10% (v/v) glycerol, and cOmplete protease inhibitor (Roche)) for 3 h. The supernatant was collected after centrifugation at 108,000 × g for 35 min and incubated with 1.2 mL of TALON beads (GE Healthcare) overnight.

Bradford Assay:

Article Title: High-level expression of the HIV entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves
Article Snippet: Total soluble protein was extracted from leaf samples homogenized in a buffer (buffer 1) containing 50 mM N -2-hydroxyethylpiperazin- N′ -2-ethanesulfonic acid (HEPES)-KOH (pH 7.5), 10 mM potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 1 mM dithiothreitol and 1 mM cOmplete protease inhibitor (Roche). .. Total soluble protein concentrations were determined by the Bradford assay (Roche, Karlsruhe, Germany) using known concentrations of bovine serum albumin (BSA) as a protein standard.

Western Blot:

Article Title: Proteome-wide covalent ligand discovery in native biological systems
Article Snippet: For western blot analysis, 10 mL of stimulated primary human T cells (1.5 million cells/mL) in RPMI with IL2 were then treated with the indicated compounds for 1h prior to addition of FasL (1 µL of 100 µg/µL stock solution of Mega FasLigand™ in water, final concentration =10 ng/mL, Adipogen). .. After 3 hours, cells were harvested by centrifugation, washed in PBS and lysed in cell lysis buffer (BioVision, 1067-100) with 1 × cOmplete protease inhibitor (Roche) and 40 µg of each sample were separated by SDS-Page on 14% polyacrylamide gels.

Article Title: Sorafenib inhibits intracellular signaling pathways and induces cell cycle arrest and cell death in thyroid carcinoma cells irrespective of histological origin or BRAF mutational status
Article Snippet: Paragraph title: Proteome Profiler™ array and western blot analysis ... Cells were lysed in lysis buffer containing cOmplete protease inhibitor and phosSTOP phosphatase inhibitor cocktails (Roche Applied Science, Mannheim, Germany).

Transformation Assay:

Article Title: Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress
Article Snippet: Cells expressing Fps1-3xFLAG (yAM271-A), Fps13A -3xFLAG (yAM272-A) or untagged Fps1 (BY4742) were transformed with empty vector or the same vector expressing Fps1-3xFLAG (pAX302) or Fps13A -3xFLAG (pAX303) under control of the MET25 promoter. .. Cells were harvested by centrifugation and resuspended in 5 ml of TNE+Triton+NP-40 (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 4 mM NaVO4 , 50 mM NaF, 20 mM Na-PPi, 5 mM EDTA, 5 mM EGTA, 0.5% Triton-X100, 1.0% NP-40, 1× cOmplete protease inhibitor [Roche, Pleasanton, California, United States]).

Article Title: Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii
Article Snippet: After sequence verification, pET-41a-His6 -MJ1099 was transformed into E. coli BL21 (DE3) RIL cells (Stratagene, La Jolla, California, USA). .. Approximately 10 g of cells were suspended 1:3( w : v ) in 50 m M Tris–HCl pH 8.0 containing 300 m M NaCl, 5% glycerol, 20 m M imidazole, 1 mg ml−1 lysozyme, 0.01 mg ml−1 DNase, 4 m M dithiothreitol (DTT) and Roche cOmplete protease inhibitor (Roche, Nutley, New Jersey, USA).

Chromatography:

Article Title: Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii
Article Snippet: Approximately 10 g of cells were suspended 1:3( w : v ) in 50 m M Tris–HCl pH 8.0 containing 300 m M NaCl, 5% glycerol, 20 m M imidazole, 1 mg ml−1 lysozyme, 0.01 mg ml−1 DNase, 4 m M dithiothreitol (DTT) and Roche cOmplete protease inhibitor (Roche, Nutley, New Jersey, USA). .. His6 -MJ1099 was purified from filtered lysate using nickel-affinity chromatography.

Immunoprecipitation:

Article Title: The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome
Article Snippet: Protein Identification by Mass Spectrometry Analysis TRIM50 complexes were isolated from HEK293 cells total extracts by immunoprecipitation. .. FLAG-TRIM50#3 and FLAG#3 cell lines were lysed in PBS, 0.5% NP-40, 1 mM PMSF, and COMPLETE protease inhibitors (Roche) for 45 min under gently mixed.

Article Title: Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress
Article Snippet: Cells were harvested by centrifugation and resuspended in 5 ml of TNE+Triton+NP-40 (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 4 mM NaVO4 , 50 mM NaF, 20 mM Na-PPi, 5 mM EDTA, 5 mM EGTA, 0.5% Triton-X100, 1.0% NP-40, 1× cOmplete protease inhibitor [Roche, Pleasanton, California, United States]). .. Protein concentration of the clarified lysate was measured using BCA reagent (Thermo Fisher Scientific, Waltham, Massachusetts, United States) and then Fps1-3xFLAG was immunoprecipitated from a volume of extract containing a total of 10 mg protein using 50 μl of mouse anti-FLAG antibody coupled-agarose resin (Sigma Aldrich) equilibrated in TNE+Triton+NP-40.

Protease Inhibitor:

Article Title: Proteome-wide covalent ligand discovery in native biological systems
Article Snippet: .. After 3 hours, cells were harvested by centrifugation, washed in PBS and lysed in cell lysis buffer (BioVision, 1067-100) with 1 × cOmplete protease inhibitor (Roche) and 40 µg of each sample were separated by SDS-Page on 14% polyacrylamide gels. ..

Article Title: The mechanism of DNA replication termination in vertebrates
Article Snippet: .. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, 1 mM DTT, 10% sucrose (w/v), cOmplete protease inhibitor (Roche, Nutley, NJ). ..

Article Title: High-level expression of the HIV entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves
Article Snippet: .. Total soluble protein was extracted from leaf samples homogenized in a buffer (buffer 1) containing 50 mM N -2-hydroxyethylpiperazin- N′ -2-ethanesulfonic acid (HEPES)-KOH (pH 7.5), 10 mM potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 1 mM dithiothreitol and 1 mM cOmplete protease inhibitor (Roche). ..

Article Title: Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress
Article Snippet: .. Cells were harvested by centrifugation and resuspended in 5 ml of TNE+Triton+NP-40 (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 4 mM NaVO4 , 50 mM NaF, 20 mM Na-PPi, 5 mM EDTA, 5 mM EGTA, 0.5% Triton-X100, 1.0% NP-40, 1× cOmplete protease inhibitor [Roche, Pleasanton, California, United States]). .. The cells were then lysed cryogenically using Mixer Mill MM301 (Retsch GmbH, Haan, Germany).

Article Title: PIP2 stabilises active states of GPCRs and enhances the selectivity of G-protein coupling
Article Snippet: .. The membranes were pelleted by ultracentrifugation at 108,000 × g for 35 min and solubilized in solubilisation buffer (50 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, 1% decyl-β-D-maltopyranoside (DM) (w/v), 10% (v/v) glycerol, and cOmplete protease inhibitor (Roche)) for 3 h. The supernatant was collected after centrifugation at 108,000 × g for 35 min and incubated with 1.2 mL of TALON beads (GE Healthcare) overnight. ..

Article Title: Sorafenib inhibits intracellular signaling pathways and induces cell cycle arrest and cell death in thyroid carcinoma cells irrespective of histological origin or BRAF mutational status
Article Snippet: .. Cells were lysed in lysis buffer containing cOmplete protease inhibitor and phosSTOP phosphatase inhibitor cocktails (Roche Applied Science, Mannheim, Germany). ..

Article Title: Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii
Article Snippet: .. Approximately 10 g of cells were suspended 1:3( w : v ) in 50 m M Tris–HCl pH 8.0 containing 300 m M NaCl, 5% glycerol, 20 m M imidazole, 1 mg ml−1 lysozyme, 0.01 mg ml−1 DNase, 4 m M dithiothreitol (DTT) and Roche cOmplete protease inhibitor (Roche, Nutley, New Jersey, USA). .. Cells were broken using an Emulsiflex homogenizer (Avestin Inc., Ottawa, Ontario, Canada) as described by Peti & Page (2007 ).

Article Title: Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria
Article Snippet: .. Cultures were harvested by centrifugation and then re-suspended in 50 mM phosphate buffer pH 7.0, 0.5 M NaCl, 1 mM dithiothreitol (DTT) and cOmplete™ protease inhibitor (Roche Diagnostics, Mannheim, Germany). ..

Article Title: Insights into ubiquitin chain architecture using Ub-clipping
Article Snippet: .. OA-treated cells were resuspended in 20 mM HEPES (pH 7.6), 220 mM mannitol, 70 mM sucrose, 1 mM EDTA, supplemented with 1x cOmplete protease inhibitor (Roche) and 1x PhosStop (Roche). .. Following homogenisation with a dounce homogeniser, samples were pelleted for 5 min at 1,000 x g, 4 °C.

Article Title: Microtubule-assisted altered trafficking of astrocytic gap junction protein connexin 43 is associated with depletion of connexin 47 during mouse hepatitis virus infection
Article Snippet: .. Following centrifugation, cells were resuspended with PBS containing 1% Triton X-100, 1 m m EDTA, 1× cOmplete protease inhibitor (Roche Applied Science), and phosphatase inhibitors (1 m m NaVO4 and 10 m m NaF) at 4 °C. .. Cells were homogenized at regular intervals, and after successful lysis in non-denaturing conditions, samples were centrifuged at 1000 × g for 5 min at 4 °C.

Article Title: PIP2 stabilises active states of GPCRs and enhances the selectivity of G-protein coupling
Article Snippet: .. The membranes were pelleted by ultracentrifugation at 108,000 × g for 35 min and solubilized in solubilisation buffer (50 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, 1% decyl-β-D-maltopyranoside (DM) (w/v), 10% (v/v) glycerol, and cOmplete protease inhibitor (Roche)) for 3 h. The supernatant was collected after centrifugation at 108,000 × g for 35 min and incubated with 1.2 mL of TALON beads (GE Healthcare) overnight. ..

Buffer Exchange:

Article Title: PIP2 stabilises active states of GPCRs and enhances the selectivity of G-protein coupling
Article Snippet: The membranes were pelleted by ultracentrifugation at 108,000 × g for 35 min and solubilized in solubilisation buffer (50 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, 1% decyl-β-D-maltopyranoside (DM) (w/v), 10% (v/v) glycerol, and cOmplete protease inhibitor (Roche)) for 3 h. The supernatant was collected after centrifugation at 108,000 × g for 35 min and incubated with 1.2 mL of TALON beads (GE Healthcare) overnight. .. Following the buffer exchange to storage buffer (20 mM HEPES pH 7, 100 mM NaCl, 0.1 mM MgCl2 , 4 mM β-mercaptoethanol, 10 % (v/)v) glycerol, and 0.5% (w/v) DM) and reverse IMAC by Ni-NTA superflow beads (GE Healthcare), G protein complex was concentrated to at least 2 mg/mL for experimental use.

Article Title: PIP2 stabilises active states of GPCRs and enhances the selectivity of G-protein coupling
Article Snippet: The membranes were pelleted by ultracentrifugation at 108,000 × g for 35 min and solubilized in solubilisation buffer (50 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, 1% decyl-β-D-maltopyranoside (DM) (w/v), 10% (v/v) glycerol, and cOmplete protease inhibitor (Roche)) for 3 h. The supernatant was collected after centrifugation at 108,000 × g for 35 min and incubated with 1.2 mL of TALON beads (GE Healthcare) overnight. .. Following the buffer exchange to storage buffer (20 mM HEPES pH 7, 100 mM NaCl, 0.1 mM MgCl2 , 4 mM β-mercaptoethanol, 10 % (v/)v) glycerol, and 0.5% (w/v) DM) and reverse IMAC by Ni-NTA superflow beads (GE Healthcare), G protein complex was concentrated to at least 2 mg/mL for experimental use.

Cell Culture:

Article Title: Proteome-wide covalent ligand discovery in native biological systems
Article Snippet: Apoptosis assays in primary human T cells with CASP8 inhibitors Primary human T cells were stimulated for 3 days with anti-CD3 and anti-CD28, and the cells were then washed and cultured in complete RPMI with IL2 (10 µg/mL) for 4 additional days. .. After 3 hours, cells were harvested by centrifugation, washed in PBS and lysed in cell lysis buffer (BioVision, 1067-100) with 1 × cOmplete protease inhibitor (Roche) and 40 µg of each sample were separated by SDS-Page on 14% polyacrylamide gels.

Protein Concentration:

Article Title: Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress
Article Snippet: Cells were harvested by centrifugation and resuspended in 5 ml of TNE+Triton+NP-40 (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 4 mM NaVO4 , 50 mM NaF, 20 mM Na-PPi, 5 mM EDTA, 5 mM EGTA, 0.5% Triton-X100, 1.0% NP-40, 1× cOmplete protease inhibitor [Roche, Pleasanton, California, United States]). .. Protein concentration of the clarified lysate was measured using BCA reagent (Thermo Fisher Scientific, Waltham, Massachusetts, United States) and then Fps1-3xFLAG was immunoprecipitated from a volume of extract containing a total of 10 mg protein using 50 μl of mouse anti-FLAG antibody coupled-agarose resin (Sigma Aldrich) equilibrated in TNE+Triton+NP-40.

Sequencing:

Article Title: Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii
Article Snippet: After sequence verification, pET-41a-His6 -MJ1099 was transformed into E. coli BL21 (DE3) RIL cells (Stratagene, La Jolla, California, USA). .. Approximately 10 g of cells were suspended 1:3( w : v ) in 50 m M Tris–HCl pH 8.0 containing 300 m M NaCl, 5% glycerol, 20 m M imidazole, 1 mg ml−1 lysozyme, 0.01 mg ml−1 DNase, 4 m M dithiothreitol (DTT) and Roche cOmplete protease inhibitor (Roche, Nutley, New Jersey, USA).

Article Title: Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria
Article Snippet: Briefly, cDNA synthesised de novo encoding amino acids 44 to 207 of the full length human CypD sequence (Uniprot P30405) without a stop codon was inserted into the Pet21a vector (Novagen) at the Nde1 and BamH1 restriction sites. .. Cultures were harvested by centrifugation and then re-suspended in 50 mM phosphate buffer pH 7.0, 0.5 M NaCl, 1 mM dithiothreitol (DTT) and cOmplete™ protease inhibitor (Roche Diagnostics, Mannheim, Germany).

Sonication:

Article Title: The mechanism of DNA replication termination in vertebrates
Article Snippet: Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, 1 mM DTT, 10% sucrose (w/v), cOmplete protease inhibitor (Roche, Nutley, NJ). .. Chromatin-bound LacR was then released by sonication (in 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 M NaCl, 1 mM DTT, cOmplete protease inhibitor, 30 mM IPTG).

Injection:

Article Title: Multiple kinases inhibit origin licensing and helicase activation to ensure reductive cell division during meiosis
Article Snippet: Cells were lysed using a freezer mill in Buffer H (25 mM Hepes [pH = 7.6], 5 mM magnesium acetate [MgAc], 1 mM EDTA, 1 mM EGTA, 10% glycerol) containing 1 M Sorbitol, 0.02% NP–40, 2 mM ATP, 0.5 M KCl, 1x cOmplete Protease Inhibitors (Roche), and PhosSTOP phosphatase inhibitors (Roche). .. Eluted protein was concentrated using a spin column (10 kDa cutoff, Vivaspin) and injected onto a Superdex 75 column (GE Healthcare) equilibrated with the same buffer.

Recombinant:

Article Title: Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria
Article Snippet: Paragraph title: Recombinant human CypD (rhCypD) protein production ... Cultures were harvested by centrifugation and then re-suspended in 50 mM phosphate buffer pH 7.0, 0.5 M NaCl, 1 mM dithiothreitol (DTT) and cOmplete™ protease inhibitor (Roche Diagnostics, Mannheim, Germany).

Molecular Weight:

Article Title: Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism
Article Snippet: Bacterial cell pellet was thawed and lysed in buffer A containing 500 mM NaCl, 25 mM Hepes, pH 7, 20 mM imidazole, cOmplete protease inhibitors (Roche), lysozyme, and benzonase. .. Fractions were collected and concentrated using Millipore 30,000 molecular weight cutoff centrifugal filters to prepare for loading on a size-exclusion chromatography (SEC) column.

Mutagenesis:

Article Title: Multiple kinases inhibit origin licensing and helicase activation to ensure reductive cell division during meiosis
Article Snippet: The Ime2–AS protein contains a M146G mutation. .. Cells were lysed using a freezer mill in Buffer H (25 mM Hepes [pH = 7.6], 5 mM magnesium acetate [MgAc], 1 mM EDTA, 1 mM EGTA, 10% glycerol) containing 1 M Sorbitol, 0.02% NP–40, 2 mM ATP, 0.5 M KCl, 1x cOmplete Protease Inhibitors (Roche), and PhosSTOP phosphatase inhibitors (Roche).

Isolation:

Article Title: The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome
Article Snippet: Protein Identification by Mass Spectrometry Analysis TRIM50 complexes were isolated from HEK293 cells total extracts by immunoprecipitation. .. FLAG-TRIM50#3 and FLAG#3 cell lines were lysed in PBS, 0.5% NP-40, 1 mM PMSF, and COMPLETE protease inhibitors (Roche) for 45 min under gently mixed.

Article Title: The mechanism of DNA replication termination in vertebrates
Article Snippet: Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, 1 mM DTT, 10% sucrose (w/v), cOmplete protease inhibitor (Roche, Nutley, NJ). .. The insoluble, chromatin-containing fraction was isolated by centrifugation at 4°C.

Article Title: High-level expression of the HIV entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves
Article Snippet: Paragraph title: Protein isolation, purification and visualization ... Total soluble protein was extracted from leaf samples homogenized in a buffer (buffer 1) containing 50 mM N -2-hydroxyethylpiperazin- N′ -2-ethanesulfonic acid (HEPES)-KOH (pH 7.5), 10 mM potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 1 mM dithiothreitol and 1 mM cOmplete protease inhibitor (Roche).

Article Title: Insights into ubiquitin chain architecture using Ub-clipping
Article Snippet: Paragraph title: Mitochondria isolation and enrichment of ubiquitinated proteins ... OA-treated cells were resuspended in 20 mM HEPES (pH 7.6), 220 mM mannitol, 70 mM sucrose, 1 mM EDTA, supplemented with 1x cOmplete protease inhibitor (Roche) and 1x PhosStop (Roche).

Size-exclusion Chromatography:

Article Title: Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism
Article Snippet: Bacterial cell pellet was thawed and lysed in buffer A containing 500 mM NaCl, 25 mM Hepes, pH 7, 20 mM imidazole, cOmplete protease inhibitors (Roche), lysozyme, and benzonase. .. Fractions were collected and concentrated using Millipore 30,000 molecular weight cutoff centrifugal filters to prepare for loading on a size-exclusion chromatography (SEC) column.

Purification:

Article Title: Multiple kinases inhibit origin licensing and helicase activation to ensure reductive cell division during meiosis
Article Snippet: Protein purifications Ime2 (strain yDP159) and Ime2–AS (strain yDP554) were purified from yeast strains containing the PGAL –IME2stable –3xFLAG construct, which expressed amino acids 1–404 of IME2 fused to a 3xFLAG epitope at the C–terminus. .. Cells were lysed using a freezer mill in Buffer H (25 mM Hepes [pH = 7.6], 5 mM magnesium acetate [MgAc], 1 mM EDTA, 1 mM EGTA, 10% glycerol) containing 1 M Sorbitol, 0.02% NP–40, 2 mM ATP, 0.5 M KCl, 1x cOmplete Protease Inhibitors (Roche), and PhosSTOP phosphatase inhibitors (Roche).

Article Title: High-level expression of the HIV entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves
Article Snippet: Paragraph title: Protein isolation, purification and visualization ... Total soluble protein was extracted from leaf samples homogenized in a buffer (buffer 1) containing 50 mM N -2-hydroxyethylpiperazin- N′ -2-ethanesulfonic acid (HEPES)-KOH (pH 7.5), 10 mM potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 1 mM dithiothreitol and 1 mM cOmplete protease inhibitor (Roche).

Article Title: PIP2 stabilises active states of GPCRs and enhances the selectivity of G-protein coupling
Article Snippet: Paragraph title: Purification of heterotrimeric G protein ... The membranes were pelleted by ultracentrifugation at 108,000 × g for 35 min and solubilized in solubilisation buffer (50 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, 1% decyl-β-D-maltopyranoside (DM) (w/v), 10% (v/v) glycerol, and cOmplete protease inhibitor (Roche)) for 3 h. The supernatant was collected after centrifugation at 108,000 × g for 35 min and incubated with 1.2 mL of TALON beads (GE Healthcare) overnight.

Article Title: Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii
Article Snippet: Paragraph title: 2.1.1. Production and purification of native His6 -MJ1099   ... Approximately 10 g of cells were suspended 1:3( w : v ) in 50 m M Tris–HCl pH 8.0 containing 300 m M NaCl, 5% glycerol, 20 m M imidazole, 1 mg ml−1 lysozyme, 0.01 mg ml−1 DNase, 4 m M dithiothreitol (DTT) and Roche cOmplete protease inhibitor (Roche, Nutley, New Jersey, USA).

Article Title: Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria
Article Snippet: Cultures were harvested by centrifugation and then re-suspended in 50 mM phosphate buffer pH 7.0, 0.5 M NaCl, 1 mM dithiothreitol (DTT) and cOmplete™ protease inhibitor (Roche Diagnostics, Mannheim, Germany). .. His-tagged rhCypD protein was purified from the supernatant using a 1 ml HisTrap FF column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) by fast protein liquid chromatography (FPLC) and then buffer exchanged into phosphate buffered saline (PBS) containing 10% glycerol and 1 mM DTT before storing at −20 °C.

Article Title: Insights into ubiquitin chain architecture using Ub-clipping
Article Snippet: OA-treated cells were resuspended in 20 mM HEPES (pH 7.6), 220 mM mannitol, 70 mM sucrose, 1 mM EDTA, supplemented with 1x cOmplete protease inhibitor (Roche) and 1x PhosStop (Roche). .. Further purification of ubiquitinated integral mitochondrial membrane proteins was achieved by sodium carbonate treatment , .

Protein Purification:

Article Title: The mechanism of DNA replication termination in vertebrates
Article Snippet: Paragraph title: Protein Purification ... Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, 1 mM DTT, 10% sucrose (w/v), cOmplete protease inhibitor (Roche, Nutley, NJ).

Article Title: Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism
Article Snippet: Paragraph title: Protein Purification. ... Bacterial cell pellet was thawed and lysed in buffer A containing 500 mM NaCl, 25 mM Hepes, pH 7, 20 mM imidazole, cOmplete protease inhibitors (Roche), lysozyme, and benzonase.

Blocking Assay:

Article Title: Sorafenib inhibits intracellular signaling pathways and induces cell cycle arrest and cell death in thyroid carcinoma cells irrespective of histological origin or BRAF mutational status
Article Snippet: Cells were lysed in lysis buffer containing cOmplete protease inhibitor and phosSTOP phosphatase inhibitor cocktails (Roche Applied Science, Mannheim, Germany). .. After blocking with 5% skim milk powder or 5% BSA in TBS, blots were incubated with the appropriate primary antibody in TBS buffer containing 0.1% Triton X-100 (TBS-T) overnight at 4°C.

Polyacrylamide Gel Electrophoresis:

Article Title: Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii
Article Snippet: Approximately 10 g of cells were suspended 1:3( w : v ) in 50 m M Tris–HCl pH 8.0 containing 300 m M NaCl, 5% glycerol, 20 m M imidazole, 1 mg ml−1 lysozyme, 0.01 mg ml−1 DNase, 4 m M dithiothreitol (DTT) and Roche cOmplete protease inhibitor (Roche, Nutley, New Jersey, USA). .. Cells were broken using an Emulsiflex homogenizer (Avestin Inc., Ottawa, Ontario, Canada) as described by Peti & Page (2007 ).

SDS Page:

Article Title: Proteome-wide covalent ligand discovery in native biological systems
Article Snippet: .. After 3 hours, cells were harvested by centrifugation, washed in PBS and lysed in cell lysis buffer (BioVision, 1067-100) with 1 × cOmplete protease inhibitor (Roche) and 40 µg of each sample were separated by SDS-Page on 14% polyacrylamide gels. ..

Article Title: The c-FLIPL Cleavage Product p43FLIP Promotes Activation of Extracellular Signal-regulated Kinase (ERK), Nuclear Factor κB (NF-κB), and Caspase-8 and T Cell Survival *
Article Snippet: Cells were washed once with ice-cold PBS and solubilized in lysis buffer (0.2% Nonidet P-40, 20 m m Tris-HCl (pH 7.4), 150 m m NaCl, 1 m m sodium orthovanadate, 10% glycerol, and cOmplete protease inhibitor (Roche Diagnostics)). .. Post-nuclear lysate proteins (20 μg/lane) were separated by 12.5% SDS-PAGE.

Article Title: Sorafenib inhibits intracellular signaling pathways and induces cell cycle arrest and cell death in thyroid carcinoma cells irrespective of histological origin or BRAF mutational status
Article Snippet: Cells were lysed in lysis buffer containing cOmplete protease inhibitor and phosSTOP phosphatase inhibitor cocktails (Roche Applied Science, Mannheim, Germany). .. For western blotting, 30 μg of total protein was denatured by boiling for 5 minutes in SDS sample buffer, then separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories).

Plasmid Preparation:

Article Title: Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress
Article Snippet: These transformants were then co-transformed with a plasmid expressing Rgc2-3xHA under control of the MET25 promoter ( ). .. Cells were harvested by centrifugation and resuspended in 5 ml of TNE+Triton+NP-40 (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 4 mM NaVO4 , 50 mM NaF, 20 mM Na-PPi, 5 mM EDTA, 5 mM EGTA, 0.5% Triton-X100, 1.0% NP-40, 1× cOmplete protease inhibitor [Roche, Pleasanton, California, United States]).

Article Title: Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii
Article Snippet: MJ1099 with an N-terminal 6×His tag (His6 -MJ1099) was codon-optimized for expression in Escherichia coli by gene synthesis (Genscript, Piscataway, New Jersey, USA) and cloned into the T7 expression vector pE-T41a (Novagen, Darmstadt, Germany) via Nde I and Hin dIII sites using standard protocols (Sambrook & Russell, 2001 ). .. Approximately 10 g of cells were suspended 1:3( w : v ) in 50 m M Tris–HCl pH 8.0 containing 300 m M NaCl, 5% glycerol, 20 m M imidazole, 1 mg ml−1 lysozyme, 0.01 mg ml−1 DNase, 4 m M dithiothreitol (DTT) and Roche cOmplete protease inhibitor (Roche, Nutley, New Jersey, USA).

Article Title: Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria
Article Snippet: Briefly, cDNA synthesised de novo encoding amino acids 44 to 207 of the full length human CypD sequence (Uniprot P30405) without a stop codon was inserted into the Pet21a vector (Novagen) at the Nde1 and BamH1 restriction sites. .. Cultures were harvested by centrifugation and then re-suspended in 50 mM phosphate buffer pH 7.0, 0.5 M NaCl, 1 mM dithiothreitol (DTT) and cOmplete™ protease inhibitor (Roche Diagnostics, Mannheim, Germany).

Positron Emission Tomography:

Article Title: Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii
Article Snippet: After sequence verification, pET-41a-His6 -MJ1099 was transformed into E. coli BL21 (DE3) RIL cells (Stratagene, La Jolla, California, USA). .. Approximately 10 g of cells were suspended 1:3( w : v ) in 50 m M Tris–HCl pH 8.0 containing 300 m M NaCl, 5% glycerol, 20 m M imidazole, 1 mg ml−1 lysozyme, 0.01 mg ml−1 DNase, 4 m M dithiothreitol (DTT) and Roche cOmplete protease inhibitor (Roche, Nutley, New Jersey, USA).

Homogenization:

Article Title: Insights into ubiquitin chain architecture using Ub-clipping
Article Snippet: OA-treated cells were resuspended in 20 mM HEPES (pH 7.6), 220 mM mannitol, 70 mM sucrose, 1 mM EDTA, supplemented with 1x cOmplete protease inhibitor (Roche) and 1x PhosStop (Roche). .. Following homogenisation with a dounce homogeniser, samples were pelleted for 5 min at 1,000 x g, 4 °C.

Produced:

Article Title: PIP2 stabilises active states of GPCRs and enhances the selectivity of G-protein coupling
Article Snippet: The membranes were pelleted by ultracentrifugation at 108,000 × g for 35 min and solubilized in solubilisation buffer (50 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, 1% decyl-β-D-maltopyranoside (DM) (w/v), 10% (v/v) glycerol, and cOmplete protease inhibitor (Roche)) for 3 h. The supernatant was collected after centrifugation at 108,000 × g for 35 min and incubated with 1.2 mL of TALON beads (GE Healthcare) overnight. .. The protein was further purified by a Superdex 200 Increase PC 3.2/300 column (GE Healthcare) and the protein tag was removed by incubation with human rhinovirus 3C protease (in-house produced) overnight.

Article Title: PIP2 stabilises active states of GPCRs and enhances the selectivity of G-protein coupling
Article Snippet: The membranes were pelleted by ultracentrifugation at 108,000 × g for 35 min and solubilized in solubilisation buffer (50 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, 1% decyl-β-D-maltopyranoside (DM) (w/v), 10% (v/v) glycerol, and cOmplete protease inhibitor (Roche)) for 3 h. The supernatant was collected after centrifugation at 108,000 × g for 35 min and incubated with 1.2 mL of TALON beads (GE Healthcare) overnight. .. The protein was further purified by a Superdex 200 Increase PC 3.2/300 column (GE Healthcare) and the protein tag was removed by incubation with human rhinovirus 3C protease (in-house produced) overnight.

Concentration Assay:

Article Title: Proteome-wide covalent ligand discovery in native biological systems
Article Snippet: For western blot analysis, 10 mL of stimulated primary human T cells (1.5 million cells/mL) in RPMI with IL2 were then treated with the indicated compounds for 1h prior to addition of FasL (1 µL of 100 µg/µL stock solution of Mega FasLigand™ in water, final concentration =10 ng/mL, Adipogen). .. After 3 hours, cells were harvested by centrifugation, washed in PBS and lysed in cell lysis buffer (BioVision, 1067-100) with 1 × cOmplete protease inhibitor (Roche) and 40 µg of each sample were separated by SDS-Page on 14% polyacrylamide gels.

Article Title: The mechanism of DNA replication termination in vertebrates
Article Snippet: Expression of LacR-Avi and the biotin ligase was induced by addition of IPTG to a final concentration of 1 mM. .. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, 1 mM DTT, 10% sucrose (w/v), cOmplete protease inhibitor (Roche, Nutley, NJ).

Article Title: Multiple kinases inhibit origin licensing and helicase activation to ensure reductive cell division during meiosis
Article Snippet: Cells were lysed using a freezer mill in Buffer H (25 mM Hepes [pH = 7.6], 5 mM magnesium acetate [MgAc], 1 mM EDTA, 1 mM EGTA, 10% glycerol) containing 1 M Sorbitol, 0.02% NP–40, 2 mM ATP, 0.5 M KCl, 1x cOmplete Protease Inhibitors (Roche), and PhosSTOP phosphatase inhibitors (Roche). .. The lysate was clarified by centrifugation at 150,000 x g. KCl concentration was adjusted to 300 mM and the lysate was clarified again by centrifugation at 25,000 x g. Lysate was incubated with 1 mL M2–resin (Sigma, A2220) and washed with Buffer H with 300 mM KGlut and 0.01% NP–40 before elution with 3xFLAG peptide.

Lysis:

Article Title: Proteome-wide covalent ligand discovery in native biological systems
Article Snippet: .. After 3 hours, cells were harvested by centrifugation, washed in PBS and lysed in cell lysis buffer (BioVision, 1067-100) with 1 × cOmplete protease inhibitor (Roche) and 40 µg of each sample were separated by SDS-Page on 14% polyacrylamide gels. ..

Article Title: The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome
Article Snippet: FLAG-TRIM50#3 and FLAG#3 cell lines were lysed in PBS, 0.5% NP-40, 1 mM PMSF, and COMPLETE protease inhibitors (Roche) for 45 min under gently mixed. .. Total protein extracts were pre-cleared with unspecific Mouse IgG Agarose Beads (Sigma) overnight in lysis buffer.

Article Title: The mechanism of DNA replication termination in vertebrates
Article Snippet: .. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, 1 mM DTT, 10% sucrose (w/v), cOmplete protease inhibitor (Roche, Nutley, NJ). ..

Article Title: PIP2 stabilises active states of GPCRs and enhances the selectivity of G-protein coupling
Article Snippet: The cells from 1L expression culture were resuspended and lysed in lysis buffer (10 mM HEPES pH 7, 20 mM KCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, and cOmplete protease inhibitor (Roche)). .. The membranes were pelleted by ultracentrifugation at 108,000 × g for 35 min and solubilized in solubilisation buffer (50 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, 1% decyl-β-D-maltopyranoside (DM) (w/v), 10% (v/v) glycerol, and cOmplete protease inhibitor (Roche)) for 3 h. The supernatant was collected after centrifugation at 108,000 × g for 35 min and incubated with 1.2 mL of TALON beads (GE Healthcare) overnight.

Article Title: The c-FLIPL Cleavage Product p43FLIP Promotes Activation of Extracellular Signal-regulated Kinase (ERK), Nuclear Factor κB (NF-κB), and Caspase-8 and T Cell Survival *
Article Snippet: .. Cells were washed once with ice-cold PBS and solubilized in lysis buffer (0.2% Nonidet P-40, 20 m m Tris-HCl (pH 7.4), 150 m m NaCl, 1 m m sodium orthovanadate, 10% glycerol, and cOmplete protease inhibitor (Roche Diagnostics)). .. Post-nuclear lysate proteins (20 μg/lane) were separated by 12.5% SDS-PAGE.

Article Title: Sorafenib inhibits intracellular signaling pathways and induces cell cycle arrest and cell death in thyroid carcinoma cells irrespective of histological origin or BRAF mutational status
Article Snippet: .. Cells were lysed in lysis buffer containing cOmplete protease inhibitor and phosSTOP phosphatase inhibitor cocktails (Roche Applied Science, Mannheim, Germany). ..

Article Title: Microtubule-assisted altered trafficking of astrocytic gap junction protein connexin 43 is associated with depletion of connexin 47 during mouse hepatitis virus infection
Article Snippet: Following centrifugation, cells were resuspended with PBS containing 1% Triton X-100, 1 m m EDTA, 1× cOmplete protease inhibitor (Roche Applied Science), and phosphatase inhibitors (1 m m NaVO4 and 10 m m NaF) at 4 °C. .. Cells were homogenized at regular intervals, and after successful lysis in non-denaturing conditions, samples were centrifuged at 1000 × g for 5 min at 4 °C.

Article Title: PIP2 stabilises active states of GPCRs and enhances the selectivity of G-protein coupling
Article Snippet: The cells from 1L expression culture were resuspended and lysed in lysis buffer (10 mM HEPES pH 7, 20 mM KCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, and cOmplete protease inhibitor (Roche)). .. The membranes were pelleted by ultracentrifugation at 108,000 × g for 35 min and solubilized in solubilisation buffer (50 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, 1% decyl-β-D-maltopyranoside (DM) (w/v), 10% (v/v) glycerol, and cOmplete protease inhibitor (Roche)) for 3 h. The supernatant was collected after centrifugation at 108,000 × g for 35 min and incubated with 1.2 mL of TALON beads (GE Healthcare) overnight.

Fast Protein Liquid Chromatography:

Article Title: Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria
Article Snippet: Cultures were harvested by centrifugation and then re-suspended in 50 mM phosphate buffer pH 7.0, 0.5 M NaCl, 1 mM dithiothreitol (DTT) and cOmplete™ protease inhibitor (Roche Diagnostics, Mannheim, Germany). .. His-tagged rhCypD protein was purified from the supernatant using a 1 ml HisTrap FF column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) by fast protein liquid chromatography (FPLC) and then buffer exchanged into phosphate buffered saline (PBS) containing 10% glycerol and 1 mM DTT before storing at −20 °C.

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  • 93
    Roche tnte buffer
    Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in <t>TNTE</t> buffer (20 mM <t>Tris,</t> pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .
    Tnte Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche deciliation medium
    A–C. Thin sections of the distal tips of Tetrahymena oral (A,B) and somatic (C) cilia. The central microtubule caps (c) link the distal tips of the central microtubules to the membrane (small arrowheads) and the distal filament caps (d) link the tips of the A-tubules of each doublet to the membrane (small arrowheads). The distal filaments (see F,H,I) at the tips of somatic cilia are thin and appear identical to those seen in Chlamydomonas flagella. The more bulbous distal filaments at the tips of oral cilia appear to be unique to Tetrahymena . D. Tetrahymena cilia purified after dibucaine <t>deciliation.</t> Cilia are intact and are completely enclosed by ciliary membranes. E. Purified ciliary membrane vesicles. F. Axoneme after demembranation with 1% NP-40. Distal filament caps at the tips of A tubules (d) and the central microtubule cap (c) crowns the tip of the central microtubules. G. Distal tip of an axoneme after extraction with MgCl 2 to release the capping structures. The tips of the A and central microtubules are intact but lack distal filaments and central microtubule caps (arrows). H,I. Negatively stained MgHSS containing central microtubule caps (c) and distal filaments (d) released from axonemes by MgCl 2 .
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    Roche complete protease inhibitors mini tablet
    Clustering of Src response kinetics. ( A ) Expression profiles for all proteins with <t>complete</t> time course data (n = 6,890) were grouped into 30 clusters, using k-means. ( B ) Gene ontology enrichment analysis of six agglomerated clusters. The enrichment p-values are indicated by asterisks (*
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    99
    Roche complete protease inhibitor
    Detection of nanobody coupling to β 1 AR. Mass spectral peaks assigned to the nanobody (Nb6B9) binding to β 1 AR to form a β 1 AR·Nb6B9 complex at an equimolar ratio are highlighted (orange) and demonstrate <t>complete</t> complex formation implying a higher affinity of the nanobody than mini-G s for β 1 AR (N=3 independent experiments).
    Complete Protease Inhibitor, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 1947 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .

    Journal: Current Biology

    Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy

    doi: 10.1016/j.cub.2017.06.021

    Figure Lengend Snippet: Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .

    Article Snippet: To inhibit deubiquitinases cells were lysed in TNTE buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 1x Complete protease inhibitor (Roche), 1x PhosSTOP (Roche)) supplemented with 20 mM N-Ethylmaleimide (NEM) prior to immunoprecipitation as described.

    Techniques: Expressing, Plasmid Preparation, Construct, Incubation, Recombinant, Immunoprecipitation, Lysis, Mass Spectrometry, Mutagenesis

    A–C. Thin sections of the distal tips of Tetrahymena oral (A,B) and somatic (C) cilia. The central microtubule caps (c) link the distal tips of the central microtubules to the membrane (small arrowheads) and the distal filament caps (d) link the tips of the A-tubules of each doublet to the membrane (small arrowheads). The distal filaments (see F,H,I) at the tips of somatic cilia are thin and appear identical to those seen in Chlamydomonas flagella. The more bulbous distal filaments at the tips of oral cilia appear to be unique to Tetrahymena . D. Tetrahymena cilia purified after dibucaine deciliation. Cilia are intact and are completely enclosed by ciliary membranes. E. Purified ciliary membrane vesicles. F. Axoneme after demembranation with 1% NP-40. Distal filament caps at the tips of A tubules (d) and the central microtubule cap (c) crowns the tip of the central microtubules. G. Distal tip of an axoneme after extraction with MgCl 2 to release the capping structures. The tips of the A and central microtubules are intact but lack distal filaments and central microtubule caps (arrows). H,I. Negatively stained MgHSS containing central microtubule caps (c) and distal filaments (d) released from axonemes by MgCl 2 .

    Journal: Methods in enzymology

    Article Title: Discovery and functional evaluation of ciliary proteins in Tetrahymena thermophila

    doi: 10.1016/B978-0-12-397944-5.00013-4

    Figure Lengend Snippet: A–C. Thin sections of the distal tips of Tetrahymena oral (A,B) and somatic (C) cilia. The central microtubule caps (c) link the distal tips of the central microtubules to the membrane (small arrowheads) and the distal filament caps (d) link the tips of the A-tubules of each doublet to the membrane (small arrowheads). The distal filaments (see F,H,I) at the tips of somatic cilia are thin and appear identical to those seen in Chlamydomonas flagella. The more bulbous distal filaments at the tips of oral cilia appear to be unique to Tetrahymena . D. Tetrahymena cilia purified after dibucaine deciliation. Cilia are intact and are completely enclosed by ciliary membranes. E. Purified ciliary membrane vesicles. F. Axoneme after demembranation with 1% NP-40. Distal filament caps at the tips of A tubules (d) and the central microtubule cap (c) crowns the tip of the central microtubules. G. Distal tip of an axoneme after extraction with MgCl 2 to release the capping structures. The tips of the A and central microtubules are intact but lack distal filaments and central microtubule caps (arrows). H,I. Negatively stained MgHSS containing central microtubule caps (c) and distal filaments (d) released from axonemes by MgCl 2 .

    Article Snippet: Collect cells by centrifugation (1700 × g 3 min; swinging bucket rotor, 50 ml conical tubes), wash once with 10 mM Tris-HCl pH 7.5 and gently suspend in 20 ml of the deciliation medium (10 mM Tris-HCl pH 7.4, 50 mM sucrose, 10 mM CaCl2 , protease inhibitors (Complete, Roche)) in a 250 ml flask.

    Techniques: Purification, Staining

    Clustering of Src response kinetics. ( A ) Expression profiles for all proteins with complete time course data (n = 6,890) were grouped into 30 clusters, using k-means. ( B ) Gene ontology enrichment analysis of six agglomerated clusters. The enrichment p-values are indicated by asterisks (*

    Journal: Wellcome Open Research

    Article Title: Proteome-wide analysis of protein abundance and turnover remodelling during oncogenic transformation of human breast epithelial cells

    doi: 10.12688/wellcomeopenres.14392.1

    Figure Lengend Snippet: Clustering of Src response kinetics. ( A ) Expression profiles for all proteins with complete time course data (n = 6,890) were grouped into 30 clusters, using k-means. ( B ) Gene ontology enrichment analysis of six agglomerated clusters. The enrichment p-values are indicated by asterisks (*

    Article Snippet: Sample preparation and Liquid chromatography tandem–mass spectrometry (LC-MS/MS) Cell pellets were lysed in a buffer containing 2% SDS, 10 mM HEPES, pH 7.4, 1 mM EDTA, 1x cOmplete protease inhibitors mini tablet (Roche) and 1x tablet phosStop (Roche), sonicated at 4C using a probe sonifier (Branson, 10% power, 30 s) and then the homogenate passed through a homogenisation filter (Qiashredder, Qiagen).

    Techniques: Expressing

    Detection of nanobody coupling to β 1 AR. Mass spectral peaks assigned to the nanobody (Nb6B9) binding to β 1 AR to form a β 1 AR·Nb6B9 complex at an equimolar ratio are highlighted (orange) and demonstrate complete complex formation implying a higher affinity of the nanobody than mini-G s for β 1 AR (N=3 independent experiments).

    Journal: Nature

    Article Title: PIP2 stabilises active states of GPCRs and enhances the selectivity of G-protein coupling

    doi: 10.1038/s41586-018-0325-6

    Figure Lengend Snippet: Detection of nanobody coupling to β 1 AR. Mass spectral peaks assigned to the nanobody (Nb6B9) binding to β 1 AR to form a β 1 AR·Nb6B9 complex at an equimolar ratio are highlighted (orange) and demonstrate complete complex formation implying a higher affinity of the nanobody than mini-G s for β 1 AR (N=3 independent experiments).

    Article Snippet: The membranes were pelleted by ultracentrifugation at 108,000 × g for 35 min and solubilized in solubilisation buffer (50 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2 , 10 μM GDP, 2 mM β-mercaptoethanol, 1% decyl-β-D-maltopyranoside (DM) (w/v), 10% (v/v) glycerol, and cOmplete protease inhibitor (Roche)) for 3 h. The supernatant was collected after centrifugation at 108,000 × g for 35 min and incubated with 1.2 mL of TALON beads (GE Healthcare) overnight.

    Techniques: Binding Assay