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New England Biolabs cacl2
Cacl2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cacl2 - by Bioz Stars, 2020-01
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Clone Assay:

Article Title: NP220 mediates silencing of unintegrated retroviral DNA
Article Snippet: Bacterial protein purification The gene fragment encoding the NP220 DNA binding domain (NP220DB, corresponding to amino acid residues 1240 – 1478 of NP220) was cloned into the pGEX-5X-3 vector. .. Cell lysates were centrifuged for 30 min at 4 °C before incubating with Glutathione Sepharose beads at 4 °C for 2 h. The beads were washed 5 times with PBS and protein was eluted by elution buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM CaCl2, and 10 μg/ml Factor Xa Protease (NEB).

Article Title: Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies
Article Snippet: Paragraph title: Cloning, Expression, and Purification of Human E-cadherin-EC1-EC2 ... Both proteins were dialyzed in TBS buffer + 20 mM CaCl2, digested with enterokinase (New England Biolabs) at 25°C, and purified again by Ni-affinity chromatography to remove all traces of the cleaved 6His-tag and any residual uncleaved protein.

Centrifugation:

Article Title: The replication initiation determinant protein (RepID) modulates replication by recruiting CUL4 to chromatin
Article Snippet: .. The pellet was resuspended with nucleus extraction buffer containing 5 mM CaCl2 and micrococcal nuclease (New England Biolabs, Cat. M0247S), vortexed and incubated at 37 °C for 5 min. Chromatin-bound fractions were collected after centrifugation at 18,000×g for 5 min at 4 °C. .. Total cell lysates and chromatin-bound proteins were immunodetected following SDS-PAGE.

Article Title: Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin
Article Snippet: Cells were collected by centrifugation at 3000 × g for 10 min and 10 g of cells were suspended in 100 ml of 100 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium chloride. .. SmChiA was loaded onto Superdex200 10/300 GL equilibrated with 50 mM Tris-HCl buffer (pH 8.0) after 10 mM dithiothreitol (DTT) reduction for 1 h at 25 °C and a portion of the collected enzyme (100 μl, 99.3 μM) was mixed with 2 μl of 100 mM CaCl2 and treated by 0.1 mg ml−1 Factor Xa protease (NEB) at 23 °C for 16 h. Digested SmChiA was reduced with 10 mM DTT for 1 h at 25 °C and further purified with Suprdex200 10/300 GL equilibrated with 100 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium chloride.

Article Title: Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay
Article Snippet: .. Cell debris and nuclei were removed by centrifugation, and the supernatant was mixed with 10 μl of 100 mM CaCl2 and 2 μl micrococcal nuclease (NEB, USA) to digest the contaminating plasmid. ..

Article Title: Genome-Wide Target Analyses of Otx2 Homeoprotein in Postnatal Cortex
Article Snippet: After four pulses of 25 s each, 5 mM CaCl2 and MNase (NEB, 3,000 U in 500 μl reaction mixture) were added and incubated for 10 min at 37°C. .. Insoluble material was removed by centrifugation at 15,000 rpm for 5 min at 4°C.

Article Title: Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay
Article Snippet: .. Cell debris and nuclei were removed by centrifugation, and the supernatant was mixed with 10 μl of 100 mM CaCl2 and 2 μl micrococcal nuclease (NEB, USA) to digest free nucleic acids for 30 min. ..

Amplification:

Article Title: Heat shock represses rRNA synthesis by inactivation of TIF-IA and lncRNA-dependent changes in nucleosome positioning
Article Snippet: Briefly, cells were fixed for 10 min with 1% formaldehyde, crosslinking was quenched with 125 mM glycine for 5 min and incubated in permeabilization buffer (150 mM sucrose, 80 mM KCl, 35 mM HEPES [pH 7.4], 5 mM K2 HPO4 , 5 mM MgCl2 and 0.5 mM CaCl2 , 0.05% L-α-lysophosphatidylcholine) for 1 min. Chromatin was digested with 50 units/ml MNase (New England Biolabs) in 150 mM sucrose, 50 mM NaCl, 50 mM Tris–HCl [pH 7.4] and 2 mM CaCl2 for 20 min at room temperature. .. DNA was amplified by PCR using a linker-specific primer and a 32 P-labeled rDNA-specific primer (−63/−36 rev).

Filtration:

Article Title: Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function
Article Snippet: MBP was cleaved off in the presence of 2 mM CaCl2 using Factor Xa (NEB) and incubation for 16 h at 4°C. .. As the last step of purification, gel filtration was performed in 25 mM HEPES, pH 7.8, containing 300 mM NaCl and 5% glycerol using Sephadex G-75 resin.

Article Title: Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies
Article Snippet: The two cell lysates were purified first by Ni-affinity chromatography and then by gel filtration using a Sephacryl 100 HR HiPrep 26/60 size exclusion column (GE Healthcare). .. Both proteins were dialyzed in TBS buffer + 20 mM CaCl2, digested with enterokinase (New England Biolabs) at 25°C, and purified again by Ni-affinity chromatography to remove all traces of the cleaved 6His-tag and any residual uncleaved protein.

Construct:

Article Title: Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies
Article Snippet: Overnight protein expression at room temperature in BL21(DE3)pLysS E .coli cells (Invitrogen) afforded large quantities of soluble protein for both constructs. .. Both proteins were dialyzed in TBS buffer + 20 mM CaCl2, digested with enterokinase (New England Biolabs) at 25°C, and purified again by Ni-affinity chromatography to remove all traces of the cleaved 6His-tag and any residual uncleaved protein.

Real-time Polymerase Chain Reaction:

Article Title: Genome-Wide Target Analyses of Otx2 Homeoprotein in Postnatal Cortex
Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qPCR) ... After four pulses of 25 s each, 5 mM CaCl2 and MNase (NEB, 3,000 U in 500 μl reaction mixture) were added and incubated for 10 min at 37°C.

Incubation:

Article Title: Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function
Article Snippet: .. MBP was cleaved off in the presence of 2 mM CaCl2 using Factor Xa (NEB) and incubation for 16 h at 4°C. ..

Article Title: Mechanism of Parkin activation by PINK1
Article Snippet: .. The UBE2D3~Ub conjugate was generated by incubating UBE2D3 (20 μM) with Hs UBE1 (20 nM) and Ub (80 μM) in ubiquitination buffer supplemented with 5 μM CaCl2 at 37°C for 10 min. To remove remaining ATP, 0.5 U of Apyrase (NEB) were added and the reaction incubated at 30°C for 30 min. .. The discharge reaction was studied by addition of 1 μM phospho-Parkin wild-type or R104A to a diluted charging reaction mixture (final UBE2D3 concentration was 9 μM).

Article Title: A licensing step links AID to transcription elongation for mutagenesis in B cells
Article Snippet: For further fractionation, nuclei were washed 1× with nuclear wash buffer (10 mM Tris pH 7.4, 2 mM MgCl2, 1× CPI), then resuspended in 400 μL of nuclear wash buffer and placed at 37 °C for 5 min. CaCl2 was added to 1 mM and DNA digested by adding Mircococcal nuclease (New England Biolabs) to 9.6 U mL−1 for 10 min. Digestion was stopped by adding EGTA to 2 mM final. .. Nuclei were then re-suspended in 700 μL of 150 mM extraction buffer (10 mM Tris pH 7.4, 140 mM NaCl, 1 mM MgCL2 , 2 mM EGTA, 0.1 % Triton X-100, 1× CPI) and incubated for 2 h on a rocker at 4 °C.

Article Title: The replication initiation determinant protein (RepID) modulates replication by recruiting CUL4 to chromatin
Article Snippet: .. The pellet was resuspended with nucleus extraction buffer containing 5 mM CaCl2 and micrococcal nuclease (New England Biolabs, Cat. M0247S), vortexed and incubated at 37 °C for 5 min. Chromatin-bound fractions were collected after centrifugation at 18,000×g for 5 min at 4 °C. .. Total cell lysates and chromatin-bound proteins were immunodetected following SDS-PAGE.

Article Title: Heat shock represses rRNA synthesis by inactivation of TIF-IA and lncRNA-dependent changes in nucleosome positioning
Article Snippet: .. Briefly, cells were fixed for 10 min with 1% formaldehyde, crosslinking was quenched with 125 mM glycine for 5 min and incubated in permeabilization buffer (150 mM sucrose, 80 mM KCl, 35 mM HEPES [pH 7.4], 5 mM K2 HPO4 , 5 mM MgCl2 and 0.5 mM CaCl2 , 0.05% L-α-lysophosphatidylcholine) for 1 min. Chromatin was digested with 50 units/ml MNase (New England Biolabs) in 150 mM sucrose, 50 mM NaCl, 50 mM Tris–HCl [pH 7.4] and 2 mM CaCl2 for 20 min at room temperature. .. After incubation for 6 h at 65°C, DNA was purified and mononucleosome-sized fragments were recovered from 2% agarose gels with QIAquick Gel Extraction Kit (QIAGEN).

Article Title: Genome-Wide Target Analyses of Otx2 Homeoprotein in Postnatal Cortex
Article Snippet: .. After four pulses of 25 s each, 5 mM CaCl2 and MNase (NEB, 3,000 U in 500 μl reaction mixture) were added and incubated for 10 min at 37°C. ..

Expressing:

Article Title: Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function
Article Snippet: Paragraph title: Construction of Expression Plasmids, Overexpression, and Purification of Recombinant PgFur Protein Variants ... MBP was cleaved off in the presence of 2 mM CaCl2 using Factor Xa (NEB) and incubation for 16 h at 4°C.

Article Title: Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies
Article Snippet: Paragraph title: Cloning, Expression, and Purification of Human E-cadherin-EC1-EC2 ... Both proteins were dialyzed in TBS buffer + 20 mM CaCl2, digested with enterokinase (New England Biolabs) at 25°C, and purified again by Ni-affinity chromatography to remove all traces of the cleaved 6His-tag and any residual uncleaved protein.

Western Blot:

Article Title: A licensing step links AID to transcription elongation for mutagenesis in B cells
Article Snippet: The nuclear pellet was resuspended in 500 μL of nuclear resuspension buffer without NP-40 and then centrifuged at 100 × g for 10 min. For nuclear–cytoplasmic fractionation, the protocol was stopped here and extracts analysed by WB. .. For further fractionation, nuclei were washed 1× with nuclear wash buffer (10 mM Tris pH 7.4, 2 mM MgCl2, 1× CPI), then resuspended in 400 μL of nuclear wash buffer and placed at 37 °C for 5 min. CaCl2 was added to 1 mM and DNA digested by adding Mircococcal nuclease (New England Biolabs) to 9.6 U mL−1 for 10 min. Digestion was stopped by adding EGTA to 2 mM final.

Article Title: The replication initiation determinant protein (RepID) modulates replication by recruiting CUL4 to chromatin
Article Snippet: Paragraph title: Chromatin fractionation, co-IP and Western blotting ... The pellet was resuspended with nucleus extraction buffer containing 5 mM CaCl2 and micrococcal nuclease (New England Biolabs, Cat. M0247S), vortexed and incubated at 37 °C for 5 min. Chromatin-bound fractions were collected after centrifugation at 18,000×g for 5 min at 4 °C.

Over Expression:

Article Title: Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function
Article Snippet: Paragraph title: Construction of Expression Plasmids, Overexpression, and Purification of Recombinant PgFur Protein Variants ... MBP was cleaved off in the presence of 2 mM CaCl2 using Factor Xa (NEB) and incubation for 16 h at 4°C.

Hybridization:

Article Title: Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay
Article Snippet: Hybridization and detection were performed with a DIG DNA labeling and detection kit (Roche Diagnostics, Germany), according to the manufacturer’s instructions. (iii) RNA assay. .. Cell debris and nuclei were removed by centrifugation, and the supernatant was mixed with 10 μl of 100 mM CaCl2 and 2 μl micrococcal nuclease (NEB, USA) to digest free nucleic acids for 30 min.

Transfection:

Article Title: Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal
Article Snippet: .. Supernatants were adjusted to 2 mM CaCl2 and 44 U of micrococcal nuclease (New England BioLabs) to digest transfected plasmid DNA for 2 h at 37°C. .. The samples were adjusted to 10 mM EDTA and 0.4% SDS, and pronase (Roche) was added to a final concentration of 400 μg/ml for 2 h at 37°C to release the encapsidated icDNA and to deproteinize the icDNA.

Chromatography:

Article Title: Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies
Article Snippet: .. Both proteins were dialyzed in TBS buffer + 20 mM CaCl2, digested with enterokinase (New England Biolabs) at 25°C, and purified again by Ni-affinity chromatography to remove all traces of the cleaved 6His-tag and any residual uncleaved protein. .. The two flow-through fractions were then collected and further purified by size exclusion chromatography with TBS + 1 mM CaCl2.

Immunoprecipitation:

Article Title: The replication initiation determinant protein (RepID) modulates replication by recruiting CUL4 to chromatin
Article Snippet: The pellet was resuspended with nucleus extraction buffer containing 5 mM CaCl2 and micrococcal nuclease (New England Biolabs, Cat. M0247S), vortexed and incubated at 37 °C for 5 min. Chromatin-bound fractions were collected after centrifugation at 18,000×g for 5 min at 4 °C. .. Chromatin-bound fractions were immunoprecipitated using an anti-FLAG antibody (Sigma, F1804) with 4 µg of antibody per sample.

Protease Inhibitor:

Article Title: The replication initiation determinant protein (RepID) modulates replication by recruiting CUL4 to chromatin
Article Snippet: Cells were harvested by centrifugation at 2700×g for 5 min at 4 °C, washed and resuspended in nucleus extraction buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1.0% Triton X-100, 1 mM EDTA pH 8.0, 1 mM EGTA, 0.1% SDS, 10% glycerol, 0.5% sodium deoxycholate, protease inhibitor cocktail and phosphatase inhibitor cocktail). .. The pellet was resuspended with nucleus extraction buffer containing 5 mM CaCl2 and micrococcal nuclease (New England Biolabs, Cat. M0247S), vortexed and incubated at 37 °C for 5 min. Chromatin-bound fractions were collected after centrifugation at 18,000×g for 5 min at 4 °C.

Article Title: Genome-Wide Target Analyses of Otx2 Homeoprotein in Postnatal Cortex
Article Snippet: Pooled cortices from 10 males and 10 females (total 20 mice) were cross-linked with 1% formaldehyde in PBS for 15 min at room temperature and ChIP was performed using the ChIP-IT High Sensitivity kit (Active Motif) per the manufacturer's instructions, with some modifications: after homogenization using a Dounce homogenizer, nuclei were washed in LB0 buffer (20 mM Tris-HCl [pH7.5], 10 mM NaCl, 1 mM EDTA, 0.2% NP-40, 1 mM PMSF) and resuspended in MNase buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 2.5 mM MgCl2 , 0.1% NP-40, 1 mM DTT, 1 mM PMSF), provided with cOmplete™ EDTA-free protease inhibitor cocktail (Roche Diagnostics). .. After four pulses of 25 s each, 5 mM CaCl2 and MNase (NEB, 3,000 U in 500 μl reaction mixture) were added and incubated for 10 min at 37°C.

Generated:

Article Title: Mechanism of Parkin activation by PINK1
Article Snippet: .. The UBE2D3~Ub conjugate was generated by incubating UBE2D3 (20 μM) with Hs UBE1 (20 nM) and Ub (80 μM) in ubiquitination buffer supplemented with 5 μM CaCl2 at 37°C for 10 min. To remove remaining ATP, 0.5 U of Apyrase (NEB) were added and the reaction incubated at 30°C for 30 min. .. The discharge reaction was studied by addition of 1 μM phospho-Parkin wild-type or R104A to a diluted charging reaction mixture (final UBE2D3 concentration was 9 μM).

DNA Labeling:

Article Title: Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay
Article Snippet: Hybridization and detection were performed with a DIG DNA labeling and detection kit (Roche Diagnostics, Germany), according to the manufacturer’s instructions. (iii) RNA assay. .. Cell debris and nuclei were removed by centrifugation, and the supernatant was mixed with 10 μl of 100 mM CaCl2 and 2 μl micrococcal nuclease (NEB, USA) to digest free nucleic acids for 30 min.

Polymerase Chain Reaction:

Article Title: Heat shock represses rRNA synthesis by inactivation of TIF-IA and lncRNA-dependent changes in nucleosome positioning
Article Snippet: Briefly, cells were fixed for 10 min with 1% formaldehyde, crosslinking was quenched with 125 mM glycine for 5 min and incubated in permeabilization buffer (150 mM sucrose, 80 mM KCl, 35 mM HEPES [pH 7.4], 5 mM K2 HPO4 , 5 mM MgCl2 and 0.5 mM CaCl2 , 0.05% L-α-lysophosphatidylcholine) for 1 min. Chromatin was digested with 50 units/ml MNase (New England Biolabs) in 150 mM sucrose, 50 mM NaCl, 50 mM Tris–HCl [pH 7.4] and 2 mM CaCl2 for 20 min at room temperature. .. DNA was amplified by PCR using a linker-specific primer and a 32 P-labeled rDNA-specific primer (−63/−36 rev).

Sonication:

Article Title: Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function
Article Snippet: To purify proteins, bacterial pellets were re-suspended in cold 25 mM HEPES, pH 7.8, containing 300 mM NaCl, lysed by sonication (UP100H Hielscher Ultrasonics, 4°C), and centrifuged (30,000 × g , 20 min, 4°C). .. MBP was cleaved off in the presence of 2 mM CaCl2 using Factor Xa (NEB) and incubation for 16 h at 4°C.

Article Title: NP220 mediates silencing of unintegrated retroviral DNA
Article Snippet: The harvested cells were resuspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% NP-40 and lysed with 0.25 mg/ml lysozeme for1 h followed by sonication. .. Cell lysates were centrifuged for 30 min at 4 °C before incubating with Glutathione Sepharose beads at 4 °C for 2 h. The beads were washed 5 times with PBS and protein was eluted by elution buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM CaCl2, and 10 μg/ml Factor Xa Protease (NEB).

Article Title: Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies
Article Snippet: Cells were lysed by sonication in TBS, pH 7.4, and 1 mM CaCl2 . .. Both proteins were dialyzed in TBS buffer + 20 mM CaCl2, digested with enterokinase (New England Biolabs) at 25°C, and purified again by Ni-affinity chromatography to remove all traces of the cleaved 6His-tag and any residual uncleaved protein.

Binding Assay:

Article Title: NP220 mediates silencing of unintegrated retroviral DNA
Article Snippet: Bacterial protein purification The gene fragment encoding the NP220 DNA binding domain (NP220DB, corresponding to amino acid residues 1240 – 1478 of NP220) was cloned into the pGEX-5X-3 vector. .. Cell lysates were centrifuged for 30 min at 4 °C before incubating with Glutathione Sepharose beads at 4 °C for 2 h. The beads were washed 5 times with PBS and protein was eluted by elution buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM CaCl2, and 10 μg/ml Factor Xa Protease (NEB).

Staining:

Article Title: Mechanism of Parkin activation by PINK1
Article Snippet: The UBE2D3~Ub conjugate was generated by incubating UBE2D3 (20 μM) with Hs UBE1 (20 nM) and Ub (80 μM) in ubiquitination buffer supplemented with 5 μM CaCl2 at 37°C for 10 min. To remove remaining ATP, 0.5 U of Apyrase (NEB) were added and the reaction incubated at 30°C for 30 min. .. Samples were resolved on a 4-12% SDS NuPAGE gradient gels (Invitrogen) and stained with Instant Blue SafeStain (Expedeon).

DNA Extraction:

Article Title: Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay
Article Snippet: (i) Intracellular HBV core DNA extraction. .. Cell debris and nuclei were removed by centrifugation, and the supernatant was mixed with 10 μl of 100 mM CaCl2 and 2 μl micrococcal nuclease (NEB, USA) to digest the contaminating plasmid.

Mutagenesis:

Article Title: Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies
Article Snippet: In both DNA constructs, the Cys9Ser mutation was also introduced. .. Both proteins were dialyzed in TBS buffer + 20 mM CaCl2, digested with enterokinase (New England Biolabs) at 25°C, and purified again by Ni-affinity chromatography to remove all traces of the cleaved 6His-tag and any residual uncleaved protein.

Isolation:

Article Title: Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal
Article Snippet: Paragraph title: Isolation of intracellular viral replicative intermediates. ... Supernatants were adjusted to 2 mM CaCl2 and 44 U of micrococcal nuclease (New England BioLabs) to digest transfected plasmid DNA for 2 h at 37°C.

Flow Cytometry:

Article Title: Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies
Article Snippet: Both proteins were dialyzed in TBS buffer + 20 mM CaCl2, digested with enterokinase (New England Biolabs) at 25°C, and purified again by Ni-affinity chromatography to remove all traces of the cleaved 6His-tag and any residual uncleaved protein. .. The two flow-through fractions were then collected and further purified by size exclusion chromatography with TBS + 1 mM CaCl2.

Purification:

Article Title: Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function
Article Snippet: Paragraph title: Construction of Expression Plasmids, Overexpression, and Purification of Recombinant PgFur Protein Variants ... MBP was cleaved off in the presence of 2 mM CaCl2 using Factor Xa (NEB) and incubation for 16 h at 4°C.

Article Title: Heat shock represses rRNA synthesis by inactivation of TIF-IA and lncRNA-dependent changes in nucleosome positioning
Article Snippet: Briefly, cells were fixed for 10 min with 1% formaldehyde, crosslinking was quenched with 125 mM glycine for 5 min and incubated in permeabilization buffer (150 mM sucrose, 80 mM KCl, 35 mM HEPES [pH 7.4], 5 mM K2 HPO4 , 5 mM MgCl2 and 0.5 mM CaCl2 , 0.05% L-α-lysophosphatidylcholine) for 1 min. Chromatin was digested with 50 units/ml MNase (New England Biolabs) in 150 mM sucrose, 50 mM NaCl, 50 mM Tris–HCl [pH 7.4] and 2 mM CaCl2 for 20 min at room temperature. .. After incubation for 6 h at 65°C, DNA was purified and mononucleosome-sized fragments were recovered from 2% agarose gels with QIAquick Gel Extraction Kit (QIAGEN).

Article Title: Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin
Article Snippet: .. SmChiA was loaded onto Superdex200 10/300 GL equilibrated with 50 mM Tris-HCl buffer (pH 8.0) after 10 mM dithiothreitol (DTT) reduction for 1 h at 25 °C and a portion of the collected enzyme (100 μl, 99.3 μM) was mixed with 2 μl of 100 mM CaCl2 and treated by 0.1 mg ml−1 Factor Xa protease (NEB) at 23 °C for 16 h. Digested SmChiA was reduced with 10 mM DTT for 1 h at 25 °C and further purified with Suprdex200 10/300 GL equilibrated with 100 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium chloride. .. Collected enzyme was separated into two tubes and mixed separately with 3 mol amounts of Cy3-maleimide (GE Healthcare) or biotin-PEAC5-maleimide (Dojindo).

Article Title: Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies
Article Snippet: .. Both proteins were dialyzed in TBS buffer + 20 mM CaCl2, digested with enterokinase (New England Biolabs) at 25°C, and purified again by Ni-affinity chromatography to remove all traces of the cleaved 6His-tag and any residual uncleaved protein. .. The two flow-through fractions were then collected and further purified by size exclusion chromatography with TBS + 1 mM CaCl2.

Protein Purification:

Article Title: NP220 mediates silencing of unintegrated retroviral DNA
Article Snippet: Paragraph title: Bacterial protein purification ... Cell lysates were centrifuged for 30 min at 4 °C before incubating with Glutathione Sepharose beads at 4 °C for 2 h. The beads were washed 5 times with PBS and protein was eluted by elution buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM CaCl2, and 10 μg/ml Factor Xa Protease (NEB).

Affinity Chromatography:

Article Title: Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function
Article Snippet: MBP was cleaved off in the presence of 2 mM CaCl2 using Factor Xa (NEB) and incubation for 16 h at 4°C. .. In order to separate the PgFur protein from the MBP protein, affinity chromatography using TALON resin (Clontech) was carried out.

Polyacrylamide Gel Electrophoresis:

Article Title: Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function
Article Snippet: Protein expression levels were analyzed using denaturing polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (Ciuraszkiewicz et al., ). .. MBP was cleaved off in the presence of 2 mM CaCl2 using Factor Xa (NEB) and incubation for 16 h at 4°C.

Lysis:

Article Title: A licensing step links AID to transcription elongation for mutagenesis in B cells
Article Snippet: Briefly, ~50 × 106 CH12 cells were collected and washed 1× with ice-cold PBS prior to re-suspension in 1 mL of Lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M Sucrose, 10 % glycerol, 0.1 % Triton X-100, 1 mM DTT, 1 × CPI). .. For further fractionation, nuclei were washed 1× with nuclear wash buffer (10 mM Tris pH 7.4, 2 mM MgCl2, 1× CPI), then resuspended in 400 μL of nuclear wash buffer and placed at 37 °C for 5 min. CaCl2 was added to 1 mM and DNA digested by adding Mircococcal nuclease (New England Biolabs) to 9.6 U mL−1 for 10 min. Digestion was stopped by adding EGTA to 2 mM final.

Article Title: NP220 mediates silencing of unintegrated retroviral DNA
Article Snippet: The harvested cells were resuspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% NP-40 and lysed with 0.25 mg/ml lysozeme for1 h followed by sonication. .. Cell lysates were centrifuged for 30 min at 4 °C before incubating with Glutathione Sepharose beads at 4 °C for 2 h. The beads were washed 5 times with PBS and protein was eluted by elution buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM CaCl2, and 10 μg/ml Factor Xa Protease (NEB).

Article Title: Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay
Article Snippet: For extraction of intracellular viral core DNA, cells from one well of a 12-well plate were lysed for 10 min at room temperature with 200 μl of lysis buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.2% NP-40. .. Cell debris and nuclei were removed by centrifugation, and the supernatant was mixed with 10 μl of 100 mM CaCl2 and 2 μl micrococcal nuclease (NEB, USA) to digest the contaminating plasmid.

Article Title: Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay
Article Snippet: To extract encapsidated RNA, cells from one well of a 12-well plate were lysed with 250 μl of lysis buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 1% NP-40. .. Cell debris and nuclei were removed by centrifugation, and the supernatant was mixed with 10 μl of 100 mM CaCl2 and 2 μl micrococcal nuclease (NEB, USA) to digest free nucleic acids for 30 min.

Mouse Assay:

Article Title: Genome-Wide Target Analyses of Otx2 Homeoprotein in Postnatal Cortex
Article Snippet: Pooled cortices from 10 males and 10 females (total 20 mice) were cross-linked with 1% formaldehyde in PBS for 15 min at room temperature and ChIP was performed using the ChIP-IT High Sensitivity kit (Active Motif) per the manufacturer's instructions, with some modifications: after homogenization using a Dounce homogenizer, nuclei were washed in LB0 buffer (20 mM Tris-HCl [pH7.5], 10 mM NaCl, 1 mM EDTA, 0.2% NP-40, 1 mM PMSF) and resuspended in MNase buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 2.5 mM MgCl2 , 0.1% NP-40, 1 mM DTT, 1 mM PMSF), provided with cOmplete™ EDTA-free protease inhibitor cocktail (Roche Diagnostics). .. After four pulses of 25 s each, 5 mM CaCl2 and MNase (NEB, 3,000 U in 500 μl reaction mixture) were added and incubated for 10 min at 37°C.

Chromatin Immunoprecipitation:

Article Title: Genome-Wide Target Analyses of Otx2 Homeoprotein in Postnatal Cortex
Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qPCR) ... After four pulses of 25 s each, 5 mM CaCl2 and MNase (NEB, 3,000 U in 500 μl reaction mixture) were added and incubated for 10 min at 37°C.

SDS Page:

Article Title: Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function
Article Snippet: Protein expression levels were analyzed using denaturing polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (Ciuraszkiewicz et al., ). .. MBP was cleaved off in the presence of 2 mM CaCl2 using Factor Xa (NEB) and incubation for 16 h at 4°C.

Article Title: The replication initiation determinant protein (RepID) modulates replication by recruiting CUL4 to chromatin
Article Snippet: The pellet was resuspended with nucleus extraction buffer containing 5 mM CaCl2 and micrococcal nuclease (New England Biolabs, Cat. M0247S), vortexed and incubated at 37 °C for 5 min. Chromatin-bound fractions were collected after centrifugation at 18,000×g for 5 min at 4 °C. .. Total cell lysates and chromatin-bound proteins were immunodetected following SDS-PAGE.

Plasmid Preparation:

Article Title: NP220 mediates silencing of unintegrated retroviral DNA
Article Snippet: Bacterial protein purification The gene fragment encoding the NP220 DNA binding domain (NP220DB, corresponding to amino acid residues 1240 – 1478 of NP220) was cloned into the pGEX-5X-3 vector. .. Cell lysates were centrifuged for 30 min at 4 °C before incubating with Glutathione Sepharose beads at 4 °C for 2 h. The beads were washed 5 times with PBS and protein was eluted by elution buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM CaCl2, and 10 μg/ml Factor Xa Protease (NEB).

Article Title: Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal
Article Snippet: .. Supernatants were adjusted to 2 mM CaCl2 and 44 U of micrococcal nuclease (New England BioLabs) to digest transfected plasmid DNA for 2 h at 37°C. .. The samples were adjusted to 10 mM EDTA and 0.4% SDS, and pronase (Roche) was added to a final concentration of 400 μg/ml for 2 h at 37°C to release the encapsidated icDNA and to deproteinize the icDNA.

Article Title: Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay
Article Snippet: .. Cell debris and nuclei were removed by centrifugation, and the supernatant was mixed with 10 μl of 100 mM CaCl2 and 2 μl micrococcal nuclease (NEB, USA) to digest the contaminating plasmid. ..

Co-Immunoprecipitation Assay:

Article Title: The replication initiation determinant protein (RepID) modulates replication by recruiting CUL4 to chromatin
Article Snippet: Paragraph title: Chromatin fractionation, co-IP and Western blotting ... The pellet was resuspended with nucleus extraction buffer containing 5 mM CaCl2 and micrococcal nuclease (New England Biolabs, Cat. M0247S), vortexed and incubated at 37 °C for 5 min. Chromatin-bound fractions were collected after centrifugation at 18,000×g for 5 min at 4 °C.

Recombinant:

Article Title: Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function
Article Snippet: Paragraph title: Construction of Expression Plasmids, Overexpression, and Purification of Recombinant PgFur Protein Variants ... MBP was cleaved off in the presence of 2 mM CaCl2 using Factor Xa (NEB) and incubation for 16 h at 4°C.

Southern Blot:

Article Title: Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay
Article Snippet: For Southern blot analysis, DNA samples were resolved in 1% agarose gels and transferred to positively charged nylon membranes (Roche Diagnostics, Germany). .. Cell debris and nuclei were removed by centrifugation, and the supernatant was mixed with 10 μl of 100 mM CaCl2 and 2 μl micrococcal nuclease (NEB, USA) to digest free nucleic acids for 30 min.

Size-exclusion Chromatography:

Article Title: Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies
Article Snippet: Both proteins were dialyzed in TBS buffer + 20 mM CaCl2, digested with enterokinase (New England Biolabs) at 25°C, and purified again by Ni-affinity chromatography to remove all traces of the cleaved 6His-tag and any residual uncleaved protein. .. The two flow-through fractions were then collected and further purified by size exclusion chromatography with TBS + 1 mM CaCl2.

Homogenization:

Article Title: Genome-Wide Target Analyses of Otx2 Homeoprotein in Postnatal Cortex
Article Snippet: Pooled cortices from 10 males and 10 females (total 20 mice) were cross-linked with 1% formaldehyde in PBS for 15 min at room temperature and ChIP was performed using the ChIP-IT High Sensitivity kit (Active Motif) per the manufacturer's instructions, with some modifications: after homogenization using a Dounce homogenizer, nuclei were washed in LB0 buffer (20 mM Tris-HCl [pH7.5], 10 mM NaCl, 1 mM EDTA, 0.2% NP-40, 1 mM PMSF) and resuspended in MNase buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 2.5 mM MgCl2 , 0.1% NP-40, 1 mM DTT, 1 mM PMSF), provided with cOmplete™ EDTA-free protease inhibitor cocktail (Roche Diagnostics). .. After four pulses of 25 s each, 5 mM CaCl2 and MNase (NEB, 3,000 U in 500 μl reaction mixture) were added and incubated for 10 min at 37°C.

Concentration Assay:

Article Title: Mechanism of Parkin activation by PINK1
Article Snippet: The UBE2D3~Ub conjugate was generated by incubating UBE2D3 (20 μM) with Hs UBE1 (20 nM) and Ub (80 μM) in ubiquitination buffer supplemented with 5 μM CaCl2 at 37°C for 10 min. To remove remaining ATP, 0.5 U of Apyrase (NEB) were added and the reaction incubated at 30°C for 30 min. .. The discharge reaction was studied by addition of 1 μM phospho-Parkin wild-type or R104A to a diluted charging reaction mixture (final UBE2D3 concentration was 9 μM).

Article Title: Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin
Article Snippet: SmChiA was loaded onto Superdex200 10/300 GL equilibrated with 50 mM Tris-HCl buffer (pH 8.0) after 10 mM dithiothreitol (DTT) reduction for 1 h at 25 °C and a portion of the collected enzyme (100 μl, 99.3 μM) was mixed with 2 μl of 100 mM CaCl2 and treated by 0.1 mg ml−1 Factor Xa protease (NEB) at 23 °C for 16 h. Digested SmChiA was reduced with 10 mM DTT for 1 h at 25 °C and further purified with Suprdex200 10/300 GL equilibrated with 100 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium chloride. .. SmChiA concentration was calculated from the absorbance at 280 nm; enzyme extinction coefficient was ε 280nm = 107,050 M−1 cm−1 .

Article Title: Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal
Article Snippet: Supernatants were adjusted to 2 mM CaCl2 and 44 U of micrococcal nuclease (New England BioLabs) to digest transfected plasmid DNA for 2 h at 37°C. .. The samples were adjusted to 10 mM EDTA and 0.4% SDS, and pronase (Roche) was added to a final concentration of 400 μg/ml for 2 h at 37°C to release the encapsidated icDNA and to deproteinize the icDNA.

Fractionation:

Article Title: A licensing step links AID to transcription elongation for mutagenesis in B cells
Article Snippet: .. For further fractionation, nuclei were washed 1× with nuclear wash buffer (10 mM Tris pH 7.4, 2 mM MgCl2, 1× CPI), then resuspended in 400 μL of nuclear wash buffer and placed at 37 °C for 5 min. CaCl2 was added to 1 mM and DNA digested by adding Mircococcal nuclease (New England Biolabs) to 9.6 U mL−1 for 10 min. Digestion was stopped by adding EGTA to 2 mM final. ..

Article Title: The replication initiation determinant protein (RepID) modulates replication by recruiting CUL4 to chromatin
Article Snippet: Paragraph title: Chromatin fractionation, co-IP and Western blotting ... The pellet was resuspended with nucleus extraction buffer containing 5 mM CaCl2 and micrococcal nuclease (New England Biolabs, Cat. M0247S), vortexed and incubated at 37 °C for 5 min. Chromatin-bound fractions were collected after centrifugation at 18,000×g for 5 min at 4 °C.

Gel Extraction:

Article Title: Heat shock represses rRNA synthesis by inactivation of TIF-IA and lncRNA-dependent changes in nucleosome positioning
Article Snippet: Briefly, cells were fixed for 10 min with 1% formaldehyde, crosslinking was quenched with 125 mM glycine for 5 min and incubated in permeabilization buffer (150 mM sucrose, 80 mM KCl, 35 mM HEPES [pH 7.4], 5 mM K2 HPO4 , 5 mM MgCl2 and 0.5 mM CaCl2 , 0.05% L-α-lysophosphatidylcholine) for 1 min. Chromatin was digested with 50 units/ml MNase (New England Biolabs) in 150 mM sucrose, 50 mM NaCl, 50 mM Tris–HCl [pH 7.4] and 2 mM CaCl2 for 20 min at room temperature. .. After incubation for 6 h at 65°C, DNA was purified and mononucleosome-sized fragments were recovered from 2% agarose gels with QIAquick Gel Extraction Kit (QIAGEN).

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    New England Biolabs o glycosidase
    O Glycosidase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dnase i
    Rate of transfer of DNA from UvrA to UvrB <t>DNase</t> I footprinting of Bca UvrA and Bca UvrB-DNA complexes. Reaction mixtures contained UvrA 2 (10 nM), ± 100 nM UvrB, 2 nM F 26 50/NDB duplex with the radiolabel on the damaged strand. The reactions were incubated at 37 °C then DNase I was added for 30 sec at RT. Then the samples were processed as described in the methods. The time indicated in the figure includes the time for DNase I digestion. Panel a , Gel image showing the transition of the WT UvrA footprint to WT UvrB footprint. The right side of the gel contains a graphic representation of the DNase I footprints observed. Panel b , Gel image showing the transition of the KRAA UvrA footprint to the WT UvrB footprint. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. Asterisks indicate the position of the band used to quantify the gels, p1. Panel c , Graphic representation of the band intensities of p1 relative to the band intensity observed in lane 4, DNase I digestion in the absence of proteins. The average of 3 independent experiments was plotted with the standard deviation at each point. Data were fit to a single or double exponential using Excel.
    Dnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Rate of transfer of DNA from UvrA to UvrB DNase I footprinting of Bca UvrA and Bca UvrB-DNA complexes. Reaction mixtures contained UvrA 2 (10 nM), ± 100 nM UvrB, 2 nM F 26 50/NDB duplex with the radiolabel on the damaged strand. The reactions were incubated at 37 °C then DNase I was added for 30 sec at RT. Then the samples were processed as described in the methods. The time indicated in the figure includes the time for DNase I digestion. Panel a , Gel image showing the transition of the WT UvrA footprint to WT UvrB footprint. The right side of the gel contains a graphic representation of the DNase I footprints observed. Panel b , Gel image showing the transition of the KRAA UvrA footprint to the WT UvrB footprint. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. Asterisks indicate the position of the band used to quantify the gels, p1. Panel c , Graphic representation of the band intensities of p1 relative to the band intensity observed in lane 4, DNase I digestion in the absence of proteins. The average of 3 independent experiments was plotted with the standard deviation at each point. Data were fit to a single or double exponential using Excel.

    Journal: DNA repair

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

    doi: 10.1016/j.dnarep.2007.11.013

    Figure Lengend Snippet: Rate of transfer of DNA from UvrA to UvrB DNase I footprinting of Bca UvrA and Bca UvrB-DNA complexes. Reaction mixtures contained UvrA 2 (10 nM), ± 100 nM UvrB, 2 nM F 26 50/NDB duplex with the radiolabel on the damaged strand. The reactions were incubated at 37 °C then DNase I was added for 30 sec at RT. Then the samples were processed as described in the methods. The time indicated in the figure includes the time for DNase I digestion. Panel a , Gel image showing the transition of the WT UvrA footprint to WT UvrB footprint. The right side of the gel contains a graphic representation of the DNase I footprints observed. Panel b , Gel image showing the transition of the KRAA UvrA footprint to the WT UvrB footprint. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. Asterisks indicate the position of the band used to quantify the gels, p1. Panel c , Graphic representation of the band intensities of p1 relative to the band intensity observed in lane 4, DNase I digestion in the absence of proteins. The average of 3 independent experiments was plotted with the standard deviation at each point. Data were fit to a single or double exponential using Excel.

    Article Snippet: After incubation at 37 °C for the indicated amount of time, DNase I (1 µL of 0.03 U/µL in 50 mM CaCl2 and 100 nM bovine serum albumin, New England BioLabs) was added and after digestion for 30 sec at room temperature the reactions were stopped by the addition of sarkosyl (0.5%) and EDTA (15 mM).

    Techniques: Footprinting, BIA-KA, Incubation, Size-exclusion Chromatography, Standard Deviation

    DNase I Footprinting DNase I footprint of the WT and KRAA UvrA proteins bound to the 50 bp F 26 50/NBD duplex with the 32 P on the 5′ end of the damaged strand. The indicated proteins were incubated with 2 nM duplex in the presence of 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 MgCl 2 , 1 mM ATP, 10 mM DTT, 100 nM bovine serum albumin for 15 min at 37 °C then DNase I treated and processed as described in the methods. Panel a , WT UvrA DNase I footprint. Panel b , KRAA UvrA DNase I footprint. Both gels are loaded in the same order. The lanes contained the following: lane 1, no protein and no DNase I; lane 2, UvrA 2 , 10 nM and no DNase I; lane 3, no protein plus DNase I; lanes 4–7, increasing concentrations of UvrA 2 , 10 – 80 nM, plus DNase I; lane 8, UvrA 2 , 10 nM, WT UvrB, 100 nM, plus DNase I; lane 9, WT UvrB, 100 nM, plus DNase I. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. The bands that were quantified for the graph in panel c are denoted by the arrows, p1 and p2. Panel c ]. For more details see the Materials and Methods.

    Journal: DNA repair

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

    doi: 10.1016/j.dnarep.2007.11.013

    Figure Lengend Snippet: DNase I Footprinting DNase I footprint of the WT and KRAA UvrA proteins bound to the 50 bp F 26 50/NBD duplex with the 32 P on the 5′ end of the damaged strand. The indicated proteins were incubated with 2 nM duplex in the presence of 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 MgCl 2 , 1 mM ATP, 10 mM DTT, 100 nM bovine serum albumin for 15 min at 37 °C then DNase I treated and processed as described in the methods. Panel a , WT UvrA DNase I footprint. Panel b , KRAA UvrA DNase I footprint. Both gels are loaded in the same order. The lanes contained the following: lane 1, no protein and no DNase I; lane 2, UvrA 2 , 10 nM and no DNase I; lane 3, no protein plus DNase I; lanes 4–7, increasing concentrations of UvrA 2 , 10 – 80 nM, plus DNase I; lane 8, UvrA 2 , 10 nM, WT UvrB, 100 nM, plus DNase I; lane 9, WT UvrB, 100 nM, plus DNase I. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. The bands that were quantified for the graph in panel c are denoted by the arrows, p1 and p2. Panel c ]. For more details see the Materials and Methods.

    Article Snippet: After incubation at 37 °C for the indicated amount of time, DNase I (1 µL of 0.03 U/µL in 50 mM CaCl2 and 100 nM bovine serum albumin, New England BioLabs) was added and after digestion for 30 sec at room temperature the reactions were stopped by the addition of sarkosyl (0.5%) and EDTA (15 mM).

    Techniques: Footprinting, Incubation