All reagents were from Sigma-Aldrich unless otherwise stated. ATP was from Roche Applied Science. For concentration determination, the ε260 value of 10300 M−1 cm−1 nt−1 was used for nonhomopolymeric oligonucleotides. DNA concentrations are expressed as those of oligo- or polynucleotide molecules (as opposed to those of constituent nt units), unless otherwise stated.
Adenosine triphosphate (atp)
Manufactured by Roche
Sourced in Germany, Switzerland, United States, Japan
ATP is a laboratory instrument used for the detection and quantification of adenosine triphosphate (ATP), a key molecule involved in energy transfer within living cells. It provides accurate and reliable measurements of ATP levels, which can be used as an indicator of biological activity or the presence of microbial contamination.
Automatically generated - may contain errors
Lab products found in correlation
Creatine kinase, by Roche (10 mentions)
Creatine phosphate, by Roche (7 mentions)
Adenosine diphosphate (adp), by Roche (4 mentions)
Lipopolysaccharides (lps), by InvivoGen (3 mentions)
Lactate dehydrogenase, by Roche (3 mentions)
Phosphoenolpyruvate, by Roche (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cd527a 1, by System Biosciences (2 mentions)
Potassium chloride (kcl), by Merck Group (2 mentions)
Adenosine diphosphate (adp), by Bio Basic (2 mentions)
Mgcl2 6h2o, by Bio Basic (2 mentions)
Amino acid, by Merck Group (2 mentions)
Creatine phosphokinase, by Roche (2 mentions)
Hepes koh, by Carl Roth (2 mentions)
Sodium acetate, by Merck Group (2 mentions)
Primestar hs dna polymerase, by Takara Bio (2 mentions)
Pcr clean up column, by Qiagen (2 mentions)
Ni nta spin kit, by Qiagen (2 mentions)
Origin software, by OriginLab (2 mentions)
L 14c u leucine, by PerkinElmer (2 mentions)
69 protocols using adenosine triphosphate (atp)
1
Oligonucleotide Concentration Determination
2
Single-Molecule TIRF Imaging of EcMutS
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Single-molecule fluorescence data were attained on a home-built prism-type TIRF microscope (26 (link)) with green (532 nm) and red (635 nm) laser lines. Emissions were split by a Dual View optical setup (DV2, Photometrics) in bypass mode before collection using an EMCCD camera (ProEM Exelon512, Princeton Instruments). Laser excitation was modulated by the opening and closing of shutter controlled by Micro-Manager image capture software (78 ).
The single-molecule imaging buffer was 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.1 mM DTT, 200 μg/ml acetylated BSA (Promega), 0.0025% P-20 surfactant (GE Healthcare), 1 mM ATP (Roche), and stated [NaCl]. The imaging buffer included saturated (∼3 mM) Trolox, PCA (1 mM), and PCD (10 nM) to minimize photo blinking and photobleaching (79 (link), 80 (link)).
EcMutS lifetime was calculated by flowing AF647-EcMutS (5 nM) into the prepared flow cell. In the absence of flow protein–DNA interactions were observed live. After recording Syto 59 (1000 nM, Invitrogen) was used to stain the DNA. At least two laser power settings were examined to clearly distinguish lifetime-dependent dissociation from fluorophore photobleaching. Reported lifetimes varied less than 10% at two laser power settings.
The single-molecule imaging buffer was 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.1 mM DTT, 200 μg/ml acetylated BSA (Promega), 0.0025% P-20 surfactant (GE Healthcare), 1 mM ATP (Roche), and stated [NaCl]. The imaging buffer included saturated (∼3 mM) Trolox, PCA (1 mM), and PCD (10 nM) to minimize photo blinking and photobleaching (79 (link), 80 (link)).
EcMutS lifetime was calculated by flowing AF647-EcMutS (5 nM) into the prepared flow cell. In the absence of flow protein–DNA interactions were observed live. After recording Syto 59 (1000 nM, Invitrogen) was used to stain the DNA. At least two laser power settings were examined to clearly distinguish lifetime-dependent dissociation from fluorophore photobleaching. Reported lifetimes varied less than 10% at two laser power settings.
3
Lentiviral Overexpression of CASP1
4
Cell Permeabilization and Nuclear Transport
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Import assays were performed as reported previously (Lowe et al., 2010 (link)). The buffers used were PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), permeabilization buffer (50 mM HEPES, 50 mM KOAc, 8 mM MgCl2, pH 7.3), and transport buffer (20 mM HEPES, 110 mM KOAc, 5 mM NaOAc, 2 mM MgOAc, 2 mM DTT, pH 7.3). The cell permeabilization protocol is based on that of Adam et al. (1990) (link). The cells were washed for 3 × 2 min with PBS, followed by a 2-min wash with permeabilization buffer, followed by a 5-min permeabilization with digitonin (Sigma–Aldrich) at a concentration of 50 μg/ml supplemented with an energy regenerating system of 100 µM ATP (Roche), 100 μM GTP (Roche), 4 mM creatine phosphate (Roche), and 20 U/ml creatine kinase (Roche) in permeabilization buffer. The digitonin was subsequently removed by washing for 3 × 3 min with transport buffer. After the final wash, excess liquid was removed and the appropriate experimental reaction mix was quickly added to the nuclei. Control experiments with fluorescently (FITC) labeled dextrans (70 kDa) were used to confirm that the nuclear envelope remained intact following the digitonin permeabilization.
5
Visualizing Stained λ-DNA Ligation
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λ-DNA (Roche) was stained with YOYO-1 (Invitrogen) at a ratio of 1 dye per 20 base pairs for 48 hours at 4 °C in 0.5× TBE buffer (pH 8). The contour length of unstained λ-DNA (48.5 kbp) is around 16 μm, which increases somewhat after staining47 (link). T4 DNA ligase (Roche) had a final concentration of 40 units/ml in a 0.5× TBE buffer (pH 8) that was modified as follows. In order to obtain catalytically active ligase, 1 mM ATP (Roche) and 5 mM MgCl2 were added as T4 DNA ligase cofactors to catalytically active solutions. This solution was incubated for 1 hour at 16 °C to allow full equilibrization of Mg2+, ATP, and T4 DNA ligase. After this incubation the free Mg2+ concentration was lowered by adding 2 mM EDTA. This is necessary since YOYO-1 is not a stable intercalator at a free Mg2+ concentration of 5 mM. Stained DNA was added to the protein yield a final concentration of 5 μg/mL followed by stabilization for 2 hours at 25 °C. Tests using alternative DNA (human genomic DNA from Roche) and an alternative enzyme provider (New England Biolabs) were conducted to exclude possible contamination. In a subset of experiments, a high concentration (0.6 mg/ml) bovine serum albumin (BSA) was added to prevent sticking to the nanochannel walls.
6
Nucleotide and Actin Binding Assays
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All chemicals and reagents were of the highest purity commercially available. ATP was purchased from Roche Applied Science (Penzberg, Germany) and ADP was purchased from Bio Basic (Markham, ON, Canada). Nucleotide concentrations were determined by absorbance at 259 nm using ε259 of 15,400 M−1 cm−1. In all experiments one molar equivalent of MgCl2 was added to nucleotide solutions immediately before use.
3-(N-Morpholino)propanesulfonic acid (MOPS), Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), Apyrase (potato grade VII), phalloidin were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Alternatively, the same form of phalloidin was purchased also from Setareh Biotech (Eugene, OR, USA). MgCl2∙6H2O came from Bio Basic (Markham, ON, Canada) and KCl from Merk (Darmstadt, Germany).
3-(N-Morpholino)propanesulfonic acid (MOPS), Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), Apyrase (potato grade VII), phalloidin were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Alternatively, the same form of phalloidin was purchased also from Setareh Biotech (Eugene, OR, USA). MgCl2∙6H2O came from Bio Basic (Markham, ON, Canada) and KCl from Merk (Darmstadt, Germany).
7
Reconstitution of Min protein dynamics
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8
Measuring ATP Levels in Activated CD8+ T Cells
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CD8+ T cells were isolated from PBMC as described above. 100,000 cells per well were plated on anti-CD3-coated 96 well U-bottom plates in activation medium (as described above) and treated with 5 μM of oligonucleotides for a total treatment time of six days without the use of a transfection reagent. Activation medium and oligonucleotides were replaced after three days. As mock control, cells were cultivated in activation medium without oligonucleotide. Six days after treatment start, cell culture medium was supplemented with ATP (SIGMA-Aldrich) at a concentration of 2 µM. After an incubation time of 30 min, residual ATP concentration in cell culture supernatants or cell-free medium was determined using the ATP Bioluminescence Assay Kit HSII (Roche) according to the manufacturer’s protocol.
9
Cell-Free Protein Synthesis Protocol
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Protein synthesis was conducted in coupled transcription/translation reactions in a final volume of 25–80 μl. Cell-free synthesis reactions were composed of 40% (v/v) translationally active CHO lysate supplemented with HEPES-KOH (pH 7.6, f.c. 30 mM, Carl Roth GmbH), sodium acetate (f.c. 100 mM, Merck), Mg(OAc)2 (f.c. 3.9 mM, Merck), KOAc (f.c. 150 mM, Merck), amino acids (complete 100 μM, Merck), spermidin (f.c. 0.25 mM; Roche), Dithiothreitol (DTT, 2.5 mM, Life technologies GmbH) and energy regenerating components including creatine phosphokinase (f.c. 0.1 mg/ml, Roche), creatine phosphate (20 mM, Roche), ATP (1.75 mM, Roche) and GTP (0.3 mM, Roche). To allow for DNA transcription during cell-free protein synthesis, 1 U/μl T7 RNA polymerase, 0.3 mM of UTP (Roche) and CTP (Roche) and 0.1 mM of the cap analogue m7G(ppp)G (Prof. Edward Darzynkiewicz, Warsaw University, Poland) were added to the reaction. Additionally, PolyG primer (f.c. 12 µM, IBA) was supplemented. To monitor the protein quantity and quality, cell-free protein synthesis reactions were supplemented with radioactive 14C-leucine (f.c. 50 μM, specific radioactivity 66.67 dpm/pmol, Perkin Elmer). Batch synthesis reactions were incubated at 30°C for 3 h at 500 rpm (Thermomixer comfort, Eppendorf).
10
In Vitro Transcription and Translation Assay
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Transcription reactions were conducted with ∼5-20 ng/μl purified PCR product, in 40 mM HEPES pH 7.4, 6 mM MgCl2, 20 mM spermidine (Sigma), 10 mM DTT, 0.5 mM ATP, 0.5 mM UTP, 0.5 mM CTP, 0.1 mM GTP (Roche), 0.5 mM CAP (NEB), 0.4-0.8 U/μL rRNasin (Promega), and 0.4 U/μL SP6 polymerase (NEB) at 37°C for 60 min (Sharma et al., 2010 (link)). In vitro translation reactions in a homemade rabbit reticulocyte (RRL) system containing 1/20 volume of transcription reaction, 0.5 μCi/μL 35S-methionine (Perkin Elmer EasyTag), nuclease-treated crude rabbit reticulocyte (Green Hectares), 20 mM HEPES, 10 mM KOH, 40 μg/mL creatine kinase (Roche), 20 μg/mL pig liver tRNA, 12 mM creatine phosphate (Roche), 1 mM ATP (Roche), 1 mM GTP (Roche), 50 mM KOAc, 2 mM MgCl2, 1 mM glutathione, 0.3 mM spermidine, and 40 μM of each amino acid except for methionine (Sigma), were at 32°C for 25 min unless otherwise indicated (Shao et al., 2013 (link), Sharma et al., 2010 (link)).
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