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Stability of exosome labelling by <t>gLuc-LA</t> in serum. (a) Time-course of stability of gLuc activity of gLuc-LA-labelled B16BL6 exosomes at 37°C in 20% <t>FBS/PBS</t> buffer. The initial gLuc activity was about 10 6 RLU/s/10 µL. (b) Time-course of exosome labelling stability of gLuc-LA at 37°C in 20% FBS/PBS buffer of 4 samples.
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Article Title: Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice

Journal: Journal of Extracellular Vesicles

doi: 10.3402/jev.v4.26238

Stability of exosome labelling by gLuc-LA in serum. (a) Time-course of stability of gLuc activity of gLuc-LA-labelled B16BL6 exosomes at 37°C in 20% FBS/PBS buffer. The initial gLuc activity was about 10 6 RLU/s/10 µL. (b) Time-course of exosome labelling stability of gLuc-LA at 37°C in 20% FBS/PBS buffer of 4 samples.
Figure Legend Snippet: Stability of exosome labelling by gLuc-LA in serum. (a) Time-course of stability of gLuc activity of gLuc-LA-labelled B16BL6 exosomes at 37°C in 20% FBS/PBS buffer. The initial gLuc activity was about 10 6 RLU/s/10 µL. (b) Time-course of exosome labelling stability of gLuc-LA at 37°C in 20% FBS/PBS buffer of 4 samples.

Techniques Used: Activity Assay

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Centrifugation:

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Amplification:

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Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
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Whole Genome Amplification:

Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
Article Snippet: The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere. .. The nuclei were incubated in the same buffer but with 5U T4 DNA ligase (Thermo Scientific) at 16°C at 50 rpm for 4.5 h, and then for 30 min at room temperature.

Polymerase Chain Reaction:

Article Title: Virtual Microfluidics for digital quantification and single-cell sequencing
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Article Title: Ribosome display for the rapid generation of high-affinity Zika-neutralizing single-chain antibodies
Article Snippet: To select specific antibody fragments the 0.5 ml PCR tubes were coated with 1 μg/ml of the recombinant ZIKV E protein in 100 μl PBS at 4° C overnight. .. Protein coated tubes were washed with PBS and blocked with 100 μl of molecular biology grade bovine serum albumin (BSA) in PBS (10 mg/ml) (New England Biolabs) for 1 hour at room temperature.

Mouse Assay:

Article Title: Ribosome display for the rapid generation of high-affinity Zika-neutralizing single-chain antibodies
Article Snippet: The PCR-generated DNA library of antibody-coding genes derived from the spleens of five mice were expressed in this lysate system. .. Protein coated tubes were washed with PBS and blocked with 100 μl of molecular biology grade bovine serum albumin (BSA) in PBS (10 mg/ml) (New England Biolabs) for 1 hour at room temperature.

Cytometry:

Article Title: Amplification of TGFβ Induced ITGB6 Gene Transcription May Promote Pulmonary Fibrosis
Article Snippet: Paragraph title: Flow Cytometry ... The cells were then labelled with 10μg anti-αvβ6 antibody in PBS for 20 minutes and an anti-mouse phycoerythrin conjugated secondary antibody (1:200 dilution; New England Biolabs, UK) for 20 minutes.

Blocking Assay:

Article Title: Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice
Article Snippet: After washing with PBS, the sample was placed in 50 mM glycine in PBS and incubated 4 times for 5 min each. .. After blocking with 5% bovine serum albumin (BSA) in PBS, rabbit anti-gLuc antibody (New England Biolabs Inc., Madison, WI, USA) diluted 1:500 was dropped onto the grid, which was then incubated for 30 min. After washing with 0.5% BSA in PBS, the sample was incubated with a 10-nm protein A-gold conjugate (BBI Solutions, Cardiff, UK) for 20 min. .. The grid was washed with PBS and transferred to drop of 1% glutaraldehyde in PBS and incubated for 5 min.

Article Title: Temperature-dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion
Article Snippet: Blocking buffer of 2% bovine serum albumin in PBS was added and incubated for 1 h at 37°C. .. After washing with PBS, wells were incubated with rabbit polyclonal antibodies raised against the amino terminus of human CFTR diluted 1:2000 in PBS (New England Biolabs, Hitchin, UK) for 1 h at 37°C and 2 h at 4°C.

Article Title: The Implication of Early Chromatin Changes in X Chromosome Inactivation
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Enzyme-linked Immunosorbent Assay:

Article Title: Temperature-dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion
Article Snippet: For the enzyme-linked immunosorbent assay (ELISA), a microtitre 96-well plate was coated with the dissolved membrane extracts in carbonate buffer (15 mmol l−1 Na2 CO3 and 35 mmol l−1 NaHCO3 , pH 9.6) and incubated overnight at 4°C. .. After washing with PBS, wells were incubated with rabbit polyclonal antibodies raised against the amino terminus of human CFTR diluted 1:2000 in PBS (New England Biolabs, Hitchin, UK) for 1 h at 37°C and 2 h at 4°C.

Incubation:

Article Title: Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice
Article Snippet: After washing with PBS, the sample was placed in 50 mM glycine in PBS and incubated 4 times for 5 min each. .. After blocking with 5% bovine serum albumin (BSA) in PBS, rabbit anti-gLuc antibody (New England Biolabs Inc., Madison, WI, USA) diluted 1:500 was dropped onto the grid, which was then incubated for 30 min. After washing with 0.5% BSA in PBS, the sample was incubated with a 10-nm protein A-gold conjugate (BBI Solutions, Cardiff, UK) for 20 min. .. The grid was washed with PBS and transferred to drop of 1% glutaraldehyde in PBS and incubated for 5 min.

Article Title: Temperature-dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion
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Expressing:

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Western Blot:

Article Title: Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking
Article Snippet: Paragraph title: In-cell/on-cell western assays ... Cells were washed with PBS+ before adding secondary Ab (α-Rabbit Dylight 800; 5151S, New England Biolabs) in 500 μl cell culture media/well at a 1:1000 dilution.

Derivative Assay:

Article Title: Ribosome display for the rapid generation of high-affinity Zika-neutralizing single-chain antibodies
Article Snippet: The PCR-generated DNA library of antibody-coding genes derived from the spleens of five mice were expressed in this lysate system. .. Protein coated tubes were washed with PBS and blocked with 100 μl of molecular biology grade bovine serum albumin (BSA) in PBS (10 mg/ml) (New England Biolabs) for 1 hour at room temperature.

Flow Cytometry:

Article Title: Amplification of TGFβ Induced ITGB6 Gene Transcription May Promote Pulmonary Fibrosis
Article Snippet: Paragraph title: Flow Cytometry ... The cells were then labelled with 10μg anti-αvβ6 antibody in PBS for 20 minutes and an anti-mouse phycoerythrin conjugated secondary antibody (1:200 dilution; New England Biolabs, UK) for 20 minutes.

Protease Inhibitor:

Article Title: Temperature-dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion
Article Snippet: The purified RBC membranes were dissolved using 50 mmol l−1 Tris–HCl (pH 7.5), 150 mmol l−1 NaCl, 1% Triton X-100 and 1% Nonidet P-40 plus protease inhibitor cocktail. .. After washing with PBS, wells were incubated with rabbit polyclonal antibodies raised against the amino terminus of human CFTR diluted 1:2000 in PBS (New England Biolabs, Hitchin, UK) for 1 h at 37°C and 2 h at 4°C.

Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
Article Snippet: Briefly, after pronuclei were isolated, they were fixed in 2% formaldehyde for 15 min and then lysed on ice in lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.5% (v/v) NP‐40 substitute (Sigma), 1% (v/v) Triton X‐100 (Sigma), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific)) for at least 15 min. .. The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere.

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Article Snippet: For treatments, seedlings were grown in the dark for 4 d and transferred to red light for 15 min. Sampled seedlings were ground in liquid nitrogen and further homogenized in a denaturing buffer (100 mM NaH2 PO4 , pH 8.0, 10 mM Tris-Cl, 8 M urea, 1 mM PMSF, and 1× complete protease inhibitor cocktail [Roche]). .. The pellets were then washed twice with PBS (8 g L−1 NaCl, 0.2 g L−1 KCl, 1.44 g L−1 Na2 HPO4 , and 0.24 g L−1 KH2 PO4 , pH 7.4) and once with buffer (NEB buffer 3: 100 mM NaCl, 50 mM Tris/HCl, 10 mM MgCl2 , and 1 mM DTT, pH 7.9).

Nick Translation:

Article Title: The Implication of Early Chromatin Changes in X Chromosome Inactivation
Article Snippet: Cells were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature and permeabilised for 5 min on ice in PBS containing 0.5%Triton X-100 and 2mM Vanadyl- ribonucleoside complex (New England Biolabs). .. Prior to FISH, samples were dehydrated through an ethanol series (80%, 95%,100% twice) and air-dried quickly.

Cell Culture:

Article Title: Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking
Article Snippet: Primary antibody (Ab) was added (α-KCNQ1; sc-20816, Santa Cruz) in 200 μl cell culture media/well at a 1:1000 dilution, and the cells incubated on a rocking platform at room temperature for 1 hour. .. Cells were washed with PBS+ before adding secondary Ab (α-Rabbit Dylight 800; 5151S, New England Biolabs) in 500 μl cell culture media/well at a 1:1000 dilution. .. During secondary Ab incubation, cells were protected from light and incubated on a rocking platform at room temperature for 1 hour.

Transmission Assay:

Article Title: Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice
Article Snippet: The mixture was applied to a carbon Formvar film-coated transmission electron microscope (TEM) grid (Alliance Biosystems, Osaka, Japan) and incubated for 20 min at room temperature. .. After blocking with 5% bovine serum albumin (BSA) in PBS, rabbit anti-gLuc antibody (New England Biolabs Inc., Madison, WI, USA) diluted 1:500 was dropped onto the grid, which was then incubated for 30 min. After washing with 0.5% BSA in PBS, the sample was incubated with a 10-nm protein A-gold conjugate (BBI Solutions, Cardiff, UK) for 20 min.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Ribosome display for the rapid generation of high-affinity Zika-neutralizing single-chain antibodies
Article Snippet: Protein coated tubes were washed with PBS and blocked with 100 μl of molecular biology grade bovine serum albumin (BSA) in PBS (10 mg/ml) (New England Biolabs) for 1 hour at room temperature. .. The translation/transcription mixture [containing the protein-ribosome-mRNA (PRM) complexes] was added to the washed and blocked protein-coated tubes and incubated on ice for 1 hour.

Injection:

Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
Article Snippet: To obtain G2‐phase data, zygotes were fixed 27 h post‐hCG injection (corresponding to about 15 h post‐fertilization) and lysed, and pronuclei were separated into different wells after SDS lysis according to their size. .. The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere.

Recombinant:

Article Title: Ribosome display for the rapid generation of high-affinity Zika-neutralizing single-chain antibodies
Article Snippet: To select specific antibody fragments the 0.5 ml PCR tubes were coated with 1 μg/ml of the recombinant ZIKV E protein in 100 μl PBS at 4° C overnight. .. Protein coated tubes were washed with PBS and blocked with 100 μl of molecular biology grade bovine serum albumin (BSA) in PBS (10 mg/ml) (New England Biolabs) for 1 hour at room temperature.

Hi-C:

Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
Article Snippet: Paragraph title: Single‐nucleus Hi‐C ... The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere.

Transmission Electron Microscopy:

Article Title: Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice
Article Snippet: The mixture was applied to a carbon Formvar film-coated transmission electron microscope (TEM) grid (Alliance Biosystems, Osaka, Japan) and incubated for 20 min at room temperature. .. After blocking with 5% bovine serum albumin (BSA) in PBS, rabbit anti-gLuc antibody (New England Biolabs Inc., Madison, WI, USA) diluted 1:500 was dropped onto the grid, which was then incubated for 30 min. After washing with 0.5% BSA in PBS, the sample was incubated with a 10-nm protein A-gold conjugate (BBI Solutions, Cardiff, UK) for 20 min.

Isolation:

Article Title: Temperature-dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion
Article Snippet: Paragraph title: Protocol 4: effect of in vitro heating on CFTR abundance in erythrocyte ghost membrane isolation ... After washing with PBS, wells were incubated with rabbit polyclonal antibodies raised against the amino terminus of human CFTR diluted 1:2000 in PBS (New England Biolabs, Hitchin, UK) for 1 h at 37°C and 2 h at 4°C.

Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
Article Snippet: Briefly, after pronuclei were isolated, they were fixed in 2% formaldehyde for 15 min and then lysed on ice in lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.5% (v/v) NP‐40 substitute (Sigma), 1% (v/v) Triton X‐100 (Sigma), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific)) for at least 15 min. .. The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere.

Immuno-Electron Microscopy:

Article Title: Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice
Article Snippet: Paragraph title: Immunoelectron microscopy ... After blocking with 5% bovine serum albumin (BSA) in PBS, rabbit anti-gLuc antibody (New England Biolabs Inc., Madison, WI, USA) diluted 1:500 was dropped onto the grid, which was then incubated for 30 min. After washing with 0.5% BSA in PBS, the sample was incubated with a 10-nm protein A-gold conjugate (BBI Solutions, Cardiff, UK) for 20 min.

Size-exclusion Chromatography:

Article Title: Virtual Microfluidics for digital quantification and single-cell sequencing
Article Snippet: A 25 µL hydrogel PCR reaction consisted of 2 U of VentR(exo-) polymerase (NEB), 1× ThermoPol Reaction Buffer (NEB), 0.4 mM dNTP (NEB), 1 µM Primers, 5 % DMSO (Sigma), 0.5 mg/mL BSA (NEB), 1.6 mg 4-arm PEG acrylate in PBS, 1.1 mg HS-PEG-SH in PBS, and λ DNA template (NEB) of various concentrations. .. A 25 µL hydrogel PCR reaction consisted of 2 U of VentR(exo-) polymerase (NEB), 1× ThermoPol Reaction Buffer (NEB), 0.4 mM dNTP (NEB), 1 µM Primers, 5 % DMSO (Sigma), 0.5 mg/mL BSA (NEB), 1.6 mg 4-arm PEG acrylate in PBS, 1.1 mg HS-PEG-SH in PBS, and λ DNA template (NEB) of various concentrations.

Microscopy:

Article Title: Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice
Article Snippet: The mixture was applied to a carbon Formvar film-coated transmission electron microscope (TEM) grid (Alliance Biosystems, Osaka, Japan) and incubated for 20 min at room temperature. .. After blocking with 5% bovine serum albumin (BSA) in PBS, rabbit anti-gLuc antibody (New England Biolabs Inc., Madison, WI, USA) diluted 1:500 was dropped onto the grid, which was then incubated for 30 min. After washing with 0.5% BSA in PBS, the sample was incubated with a 10-nm protein A-gold conjugate (BBI Solutions, Cardiff, UK) for 20 min.

Article Title: A Role of TGFss1 Dependent 14-3-3? Phosphorylation at Ser69 and Ser74 in the Regulation of Gene Transcription, Stemness and Radioresistance
Article Snippet: Paragraph title: Immunofluorescent microscopy ... The cells were washed with PBS and blocked with 10% BSA in PBS for 1 h. The cells were then incubated with primary antibody diluted in 3% BSA in PBS (anti-Smad3, New England Biolabs, dilution 1∶100) and anti- phospho-histon H2AXS139 (Upstate, Hamburg, Germany, dilution 1∶100) overnight at 4°C and washed ten times with PBS.

Purification:

Article Title: Virtual Microfluidics for digital quantification and single-cell sequencing
Article Snippet: In-gel PCR The primers ( ) used for PCR on purified λ DNA (48 kbp, NEB) were ordered through IDT. .. A 25 µL hydrogel PCR reaction consisted of 2 U of VentR(exo-) polymerase (NEB), 1× ThermoPol Reaction Buffer (NEB), 0.4 mM dNTP (NEB), 1 µM Primers, 5 % DMSO (Sigma), 0.5 mg/mL BSA (NEB), 1.6 mg 4-arm PEG acrylate in PBS, 1.1 mg HS-PEG-SH in PBS, and λ DNA template (NEB) of various concentrations.

Article Title: Temperature-dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion
Article Snippet: The purified RBC membranes were dissolved using 50 mmol l−1 Tris–HCl (pH 7.5), 150 mmol l−1 NaCl, 1% Triton X-100 and 1% Nonidet P-40 plus protease inhibitor cocktail. .. After washing with PBS, wells were incubated with rabbit polyclonal antibodies raised against the amino terminus of human CFTR diluted 1:2000 in PBS (New England Biolabs, Hitchin, UK) for 1 h at 37°C and 2 h at 4°C.

Article Title: Ribosome display for the rapid generation of high-affinity Zika-neutralizing single-chain antibodies
Article Snippet: Protein coated tubes were washed with PBS and blocked with 100 μl of molecular biology grade bovine serum albumin (BSA) in PBS (10 mg/ml) (New England Biolabs) for 1 hour at room temperature. .. Protein coated tubes were washed with PBS and blocked with 100 μl of molecular biology grade bovine serum albumin (BSA) in PBS (10 mg/ml) (New England Biolabs) for 1 hour at room temperature.

Article Title: Epidermal Phytochrome B Inhibits Hypocotyl Negative Gravitropism Non-Cell-Autonomously
Article Snippet: After centrifugation at 20,676 g for 10 min at 4°C, PIF3 was purified from the supernatants using Ni-NTA beads (Qiagen). .. The pellets were then washed twice with PBS (8 g L−1 NaCl, 0.2 g L−1 KCl, 1.44 g L−1 Na2 HPO4 , and 0.24 g L−1 KH2 PO4 , pH 7.4) and once with buffer (NEB buffer 3: 100 mM NaCl, 50 mM Tris/HCl, 10 mM MgCl2 , and 1 mM DTT, pH 7.9).

Sequencing:

Article Title: Interaction of amyloid-β (Aβ) oligomers with neurexin 2α and neuroligin 1 mediates synapse damage and memory loss in mice
Article Snippet: Briefly, wells coated previously with non-aggregated Aβ1–40 (prepared as described above) were blocked in 0.5% bovine serum albumin in PBS for 1 h, and 10 μl of a commercially available phage display library (PhD-C7C, New England Biolabs, Ipswich, MA) was added to each well. .. Among 24 different phages bound to Aβ, one expressed the amino acid sequence IGTVDRS displayed on the phage filamentous protein.

Labeling:

Article Title: The Implication of Early Chromatin Changes in X Chromosome Inactivation
Article Snippet: Cells were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature and permeabilised for 5 min on ice in PBS containing 0.5%Triton X-100 and 2mM Vanadyl- ribonucleoside complex (New England Biolabs). .. Prior to FISH, samples were dehydrated through an ethanol series (80%, 95%,100% twice) and air-dried quickly.

Staining:

Article Title: Virtual Microfluidics for digital quantification and single-cell sequencing
Article Snippet: A 25 µL hydrogel PCR reaction consisted of 2 U of VentR(exo-) polymerase (NEB), 1× ThermoPol Reaction Buffer (NEB), 0.4 mM dNTP (NEB), 1 µM Primers, 5 % DMSO (Sigma), 0.5 mg/mL BSA (NEB), 1.6 mg 4-arm PEG acrylate in PBS, 1.1 mg HS-PEG-SH in PBS, and λ DNA template (NEB) of various concentrations. .. The following thermal protocol was ran on an MJ Research PTC-100 twin tower thermal cycler: 30 °C for 30 min (gel polymerization), 98 °C for 3 min; 98 °C for 30 sec, 57 °C for 30 sec, 72 °C for 1 min for 40 to 60 cycles; 72 °C for 5 min and hold at 4 °C.

Article Title: A Role of TGFss1 Dependent 14-3-3? Phosphorylation at Ser69 and Ser74 in the Regulation of Gene Transcription, Stemness and Radioresistance
Article Snippet: The cells were washed with PBS and blocked with 10% BSA in PBS for 1 h. The cells were then incubated with primary antibody diluted in 3% BSA in PBS (anti-Smad3, New England Biolabs, dilution 1∶100) and anti- phospho-histon H2AXS139 (Upstate, Hamburg, Germany, dilution 1∶100) overnight at 4°C and washed ten times with PBS. .. Cells were then incubated for 1 h with a secondary antibody conjugated with Alexa 488 or 555 (Invitrogen) diluted 1∶500 in 3% BSA in PBS.

In-Cell ELISA:

Article Title: Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking
Article Snippet: For the in-cell western assay, cells were washed with PBS+ (PBS supplemented with 1 mM MgCl2 & 0.1 mM CaCl2 ) then fixed using 3.7% formaldehyde solution (PBS+ + formaldehyde) for 20 minutes. .. Cells were washed with PBS+ before adding secondary Ab (α-Rabbit Dylight 800; 5151S, New England Biolabs) in 500 μl cell culture media/well at a 1:1000 dilution.

Plasmid Preparation:

Article Title: Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking
Article Snippet: For control wells, and those destined for the addition of wortmannin (1232, Tocris Bioscience) or brefeldin A (B7651, Sigma Aldrich), 1 μg pcDNA3.1 (empty vector) was co-transfected with VSV-E1-Q1. .. Cells were washed with PBS+ before adding secondary Ab (α-Rabbit Dylight 800; 5151S, New England Biolabs) in 500 μl cell culture media/well at a 1:1000 dilution.

In Vitro:

Article Title: Temperature-dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion
Article Snippet: Paragraph title: Protocol 4: effect of in vitro heating on CFTR abundance in erythrocyte ghost membrane isolation ... After washing with PBS, wells were incubated with rabbit polyclonal antibodies raised against the amino terminus of human CFTR diluted 1:2000 in PBS (New England Biolabs, Hitchin, UK) for 1 h at 37°C and 2 h at 4°C.

Article Title: Ribosome display for the rapid generation of high-affinity Zika-neutralizing single-chain antibodies
Article Snippet: In vitro transcription and translation reaction was based on a coupled rabbit reticulocyte lysate system (Promega’s TNT quick-coupled transcription-translation system) and performed according to the supplier’s protocol. .. Protein coated tubes were washed with PBS and blocked with 100 μl of molecular biology grade bovine serum albumin (BSA) in PBS (10 mg/ml) (New England Biolabs) for 1 hour at room temperature.

Negative Control:

Article Title: Temperature-dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion
Article Snippet: After washing with PBS, wells were incubated with rabbit polyclonal antibodies raised against the amino terminus of human CFTR diluted 1:2000 in PBS (New England Biolabs, Hitchin, UK) for 1 h at 37°C and 2 h at 4°C. .. After washing with PBS, wells were incubated with rabbit polyclonal antibodies raised against the amino terminus of human CFTR diluted 1:2000 in PBS (New England Biolabs, Hitchin, UK) for 1 h at 37°C and 2 h at 4°C.

Binding Assay:

Article Title: Amplification of TGFβ Induced ITGB6 Gene Transcription May Promote Pulmonary Fibrosis
Article Snippet: Non-specific binding of anti-αvβ6 antibodies (clone 6.3G9; Biogen Idec, USA) was blocked by incubating iHBECs (100,000 cells) with 5% goat serum (Sigma-Aldrich, UK) for 20 minutes. .. The cells were then labelled with 10μg anti-αvβ6 antibody in PBS for 20 minutes and an anti-mouse phycoerythrin conjugated secondary antibody (1:200 dilution; New England Biolabs, UK) for 20 minutes.

Agarose Gel Electrophoresis:

Article Title: Ribosome display for the rapid generation of high-affinity Zika-neutralizing single-chain antibodies
Article Snippet: Protein coated tubes were washed with PBS and blocked with 100 μl of molecular biology grade bovine serum albumin (BSA) in PBS (10 mg/ml) (New England Biolabs) for 1 hour at room temperature. .. The obtained cDNA was amplified in a 25 μl PCR mixture for 35 cycles of 30 s at 94°C, 30 s at 65°C, and 1 min at 72°C, and 10 mins at 72°C with MKR and KzStrep II using GoTaq DNA polymerase (Promega, USA).

In Situ:

Article Title: Ribosome display for the rapid generation of high-affinity Zika-neutralizing single-chain antibodies
Article Snippet: Protein coated tubes were washed with PBS and blocked with 100 μl of molecular biology grade bovine serum albumin (BSA) in PBS (10 mg/ml) (New England Biolabs) for 1 hour at room temperature. .. The translation/transcription mixture [containing the protein-ribosome-mRNA (PRM) complexes] was added to the washed and blocked protein-coated tubes and incubated on ice for 1 hour.

Acid Assay:

Article Title: Temperature-dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion
Article Snippet: After washing with PBS, wells were incubated with rabbit polyclonal antibodies raised against the amino terminus of human CFTR diluted 1:2000 in PBS (New England Biolabs, Hitchin, UK) for 1 h at 37°C and 2 h at 4°C. .. Bovine serum albumin alone was used as a negative control to test the specificity of the CFTR antibody.

BAC Assay:

Article Title: The Implication of Early Chromatin Changes in X Chromosome Inactivation
Article Snippet: Cells were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature and permeabilised for 5 min on ice in PBS containing 0.5%Triton X-100 and 2mM Vanadyl- ribonucleoside complex (New England Biolabs). .. Prior to FISH, samples were dehydrated through an ethanol series (80%, 95%,100% twice) and air-dried quickly.

Lysis:

Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
Article Snippet: Briefly, after pronuclei were isolated, they were fixed in 2% formaldehyde for 15 min and then lysed on ice in lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.5% (v/v) NP‐40 substitute (Sigma), 1% (v/v) Triton X‐100 (Sigma), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific)) for at least 15 min. .. The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere.

Fluorescence In Situ Hybridization:

Article Title: The Implication of Early Chromatin Changes in X Chromosome Inactivation
Article Snippet: Paragraph title: IF with RNA FISH ... Cells were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature and permeabilised for 5 min on ice in PBS containing 0.5%Triton X-100 and 2mM Vanadyl- ribonucleoside complex (New England Biolabs).

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    New England Biolabs pbs
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