Equilibrium Rho-RNA dissociation constants were determined using a filter-binding assay, as described previously (29 (link),30 (link)). Briefly, ∼10 fmoles of 32P-labeled RNA substrate were mixed with various amounts of Rho in 100 μl of binding buffer (20 mM HEPES, pH 7.5, 0.1 mM EDTA, 0.5 mM DTT, 150 mM potassium acetate and 20 μg/ml BSA) containing 0 or 1 mM ATP (or AMP-PNP). After incubation for 10 min at 30°C, the samples were filtered through stacked [top] nitrocellulose (Amersham Protran) and [bottom] cationic nylon (Pall Biodyne B) membranes using a Bio-dot SF apparatus (Biorad). No significant differences were observed in control experiments performed with 30 min incubation times (not shown). The fractions of free and Rho-bound RNA (retained on the nylon and nitrocellulose membranes, respectively) as a function of Rho concentration were then determined by phosphorimaging of the membranes using a Typhoon-Trio imager and dedicated ImageQuant TL v8.1 software (GE healthcare).
Bio dot sf apparatus
Manufactured by Bio-Rad
Sourced in Germany
The Bio-Dot SF apparatus is a versatile laboratory equipment designed for sample preparation and analysis. It enables the rapid and efficient blotting of biological samples onto various membranes, such as nitrocellulose or PVDF, using a vacuum-based system. The Bio-Dot SF apparatus provides a consistent and reproducible method for sample application, making it a valuable tool for various applications in the life sciences and biomedical research fields.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p membrane, by Merck Group (3 mentions)
Typhoon fla 7000, by GE Healthcare (2 mentions)
Immobilon ny nylon membrane, by Merck Group (2 mentions)
Imagequant las 4000 biomolecular imager, by GE Healthcare (2 mentions)
Imagequant tl v8, by GE Healthcare (1 mentions)
Typhoon trio imager, by GE Healthcare (1 mentions)
Microspin g 25 column, by GE Healthcare (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Ab9049, by Abcam (1 mentions)
Ab9050, by Cell Signaling Technology (1 mentions)
Ab1791, by Abcam (1 mentions)
Monoclonal anti flag m2, by Merck Group (1 mentions)
Immobilon p membrane, by Bio-Rad (1 mentions)
Horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Ecl system, by Cytiva (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Odyssey fluorescent imaging system, by LI COR (1 mentions)
Rnase h, by New England Biolabs (1 mentions)
Methylene blue, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
14 protocols using bio dot sf apparatus
1
Rho-RNA Dissociation Kinetics by Filter Binding
2
Quantitative Binding Assay for DNA-Protein Interactions
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DNA (20 pmol) was 5′-labelled with 25 µCi [γ-32P] ATP by T4 polynucleotide kinase (NEB, Ipswich, UK). G4 and G4mut structures were purified by 7 % SDS-PAGE. Control DNA (ds, bubble, fork, 4 fork) was purified using illustra MicroSpin G-25 columns (GE Healthcare).
DNA–protein binding was analyzed by double-filter-binding assays [35 (link)] using a 96-well Bio-Dot SF apparatus (Bio-Rad, Hercules, CA, USA) and 10 nM DNA in binding buffer (50 mM Tris/HCl pH 8.0, 125 mM KCl, 5 mM DTT, 10% (v/v) glycerol [13 (link)]). Protein concentrations increased from 0 to 65 µM Slx9. After incubation on ice for 30 min, the reactions were filtered through a nitrocellulose and a positively charged nylon membrane, followed by three washing steps with binding buffer with no glycerol. The membranes were dried and analyzed by phosphorimaging on a Typhoon FLA 7000 (GE Healthcare). Percentage values of bound Slx9 were determined using ImageQuant, and were used to obtain dissociation equilibrium constants (Kd) by curve fitting using nonlinear regression (GraphPad Prism, San Diego, CA, USA).
DNA–protein binding was analyzed by double-filter-binding assays [35 (link)] using a 96-well Bio-Dot SF apparatus (Bio-Rad, Hercules, CA, USA) and 10 nM DNA in binding buffer (50 mM Tris/HCl pH 8.0, 125 mM KCl, 5 mM DTT, 10% (v/v) glycerol [13 (link)]). Protein concentrations increased from 0 to 65 µM Slx9. After incubation on ice for 30 min, the reactions were filtered through a nitrocellulose and a positively charged nylon membrane, followed by three washing steps with binding buffer with no glycerol. The membranes were dried and analyzed by phosphorimaging on a Typhoon FLA 7000 (GE Healthcare). Percentage values of bound Slx9 were determined using ImageQuant, and were used to obtain dissociation equilibrium constants (Kd) by curve fitting using nonlinear regression (GraphPad Prism, San Diego, CA, USA).
3
Quantifying Zuo1 Binding Kinetics
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20 pmol DNA was 5′-labeled with 25 µCi [γ-32P] ATP using T4 polynucleotide kinase (NEB). G4 and G4mut structures were purified by 7% SDS-PAGE. Control DNA (ds, bubble, fork, 4 fork) was purified using illustra MicroSpin G-25 columns. DNA-protein-binding was analyzed by double-filter binding assays82 (link) using a 96-well Bio-Dot SF apparatus (Bio-Rad) and 10 nM DNA in binding buffer (50 mM Tris HCl pH 8.0, 125 mM KCl, 5 mM DTT, 10% (v/v) glycerol)81 (link). Protein concentrations increased from 0 to 20 µM Zuo1. After incubation on ice for 30 min the reactions were filtered through a nitrocellulose and a positively charged nylon membrane, followed by three washing steps with binding buffer with no glycerol. The membranes were dried and analyzed by phosphoimaging on a Typhoon FLA 7000 (GE Healthcare). Percentage values of bound Zuo1 were determined using ImageQuant and were used to obtain dissociation equilibrium constants (apparent Kd) by curve fitting using nonlinear regression (Prism, Graphpad). The sequences of oligonucleotides used in these studies are listed in Supplementary Table S3 .
4
Syndecan-1 Detection in Infected Cells
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Cell culture supernatants of infected or mock-treated HCE cells were vacuum filtered through Immobilon-Ny+ nylon membrane (Millipore Corporation, Billerica, MA) using Bio-Dot SF apparatus (Bio-Rad, Hercules, CA). Membranes were blotted overnight with antibody specific for Syndecan-1 (Santa Cruz Biotechnology, sc-6532) followed by species-specific secondary antibody and chemiluminescence detection using ImageQuant LAS 4000 biomolecular imager (GE Healthcare Life Sciences), as described above.
5
Antibody Validation for H3K36 Modifications
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Anti-H3 (active motif 39163 lot no. 26311003, Abcam ab1791). Anti-H4 (millipore 05–858 lot no. 2020541). Anti-FLAG M2 monoclonal (Sigma F1804), anti-tubulin (TAT1 kind gift from Keith Gull) (Woods et al., 1989 (link)). Anti-H3K36me2 : Abcam ab9049, Anti-H3K36me3 : Abcam ab9050, Cell signaling technology 4909. Anti-H3K36Ac (Abcam ab177179, rabbit monoclonal), Abnova (PAB31320), Rockland (600–401-I89), and Thermo Fischer (MA5-24672).
Peptide sequences used for assessment of K36 methyl Abs are listed inSupplementary file 2 .
Slot blots used a twofold serial dilution series with 150 µL of 50, 25, 12.5, 6.25, 3.125, and 1.6 µM peptides spotted on activated PVDF 0.2-micron membrane using a 48-well BioRad Bio-Dot SF apparatus. Spots were air dried for 5 min before staining with Ponceau S stain to verify equal peptide loading. The membrane was blocked in 5% BSA in TBST at RT for 1 hr, incubated with primary Ab for 1 hr at room temperature, washed with TBST, incubated with HRP-conjugated anti-rabbit secondary Ab for 30 min, washed with TBST and then developed with enhanced chemiluminescence and images captured by LI-COR imaging. Anti-H3 K36 methylation Abs used are listed above in "Antibodies used".
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Peptide sequences used for assessment of K36 methyl Abs are listed in
Slot blots used a twofold serial dilution series with 150 µL of 50, 25, 12.5, 6.25, 3.125, and 1.6 µM peptides spotted on activated PVDF 0.2-micron membrane using a 48-well BioRad Bio-Dot SF apparatus. Spots were air dried for 5 min before staining with Ponceau S stain to verify equal peptide loading. The membrane was blocked in 5% BSA in TBST at RT for 1 hr, incubated with primary Ab for 1 hr at room temperature, washed with TBST, incubated with HRP-conjugated anti-rabbit secondary Ab for 30 min, washed with TBST and then developed with enhanced chemiluminescence and images captured by LI-COR imaging. Anti-H3 K36 methylation Abs used are listed above in "Antibodies used".
6
Dot Blot Analysis of in vitro Kinase Reactions
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Dot blot analyses were carried out according to standard protocols, using Immobilon-P membrane and the BioDot SF apparatus (Bio-Rad, Munich/Germany). Thirty μl of the in vitro kinase reaction products described above or 20 μg of each EPIYA peptide were mixed in 1 mL of transfer buffer (192 mM glycine, 25 mM Tris-HCl, 20 % methanol, 0.1 % SDS, pH 8.3). Subsequently, the samples were spotted onto the Immobilon-P membranes (Merck Millipore, Darmstadt/Germany). After drying, the Dotblots were incubated with the various antibodies as described below for the Western blots.
7
Quantitative DNA Methylation Analysis
8
Quantification of Genomic 5-Methylcytosine
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Genomic DNA of RAW 264.7 was extracted by DNA extraction buffer (50 μl TE buffer, 450 μl STE buffer,10 μl 20% SDS,10 μl protein K (10 mg/ml)). The DNA samples (2 μg per dot) were spotted on a nitrocellulose membrane in a Bio-Dot SF apparatus (Bio-Rad). The membrane was baked at 80 °C for 2 h, blocked in 5% skimmed milk in TBS containing 0.1% Tween 20, and then incubated with 1:1000 dilution of 5-meC antibody (Beyotime, AF5722) overnight at 4 °C, followed by its detection using secondary antibody. Odyssey Fluorescent imaging system (LI-COR Biosciences) was used for visualization. For the staining of total DNA, the blot membrane was hybridized with 0.2% methylene blue (Leagene, DZ0094).
9
Syndecan-1 Detection in Infected Cells
10
Peptide Dotblot Assay Protocol
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Twenty μg of each CagA peptide were mixed in 1 mL of TBST blotting buffer (140 mM NaCl; 25 mM Tris-HCl, pH 7.4; 0.1% Tween-20). These peptide samples were spotted onto Immobilon-P membrane (Merck Millipore, Darmstadt, Germany) using the BioDot SF apparatus (Bio-Rad, Munich, Germany). The resulting Dotblots were dried and subjected to antibody detection as described below for Western blots [57 (link)].
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