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.
Creatine phosphate
Manufactured by Roche
Sourced in Japan
Creatine phosphate is a laboratory reagent used in biochemical analysis. It serves as a source of high-energy phosphate groups, which are important for various cellular processes. Creatine phosphate is commonly used in assays to measure the activity of enzymes involved in energy metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Creatine kinase, by Roche (16 mentions)
Adenosine triphosphate (atp), by Roche (7 mentions)
Creatine phosphokinase, by Roche (6 mentions)
Amino acids, by Promega (4 mentions)
Digitonin, by Merck Group (2 mentions)
Amino acid mix, by Promega (2 mentions)
Amino acid, by Merck Group (2 mentions)
Hepes koh, by Carl Roth (2 mentions)
Sodium acetate, by Merck Group (2 mentions)
Creatine kinase, by Merck Group (2 mentions)
Edta free protease inhibitor cocktail, by Roche (2 mentions)
Murine rnase inhibitor, by Promega (2 mentions)
Mg oac 2, by Promega (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Micrococcal nuclease, by New England Biolabs (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Luciferase rna, by Promega (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
T7 rna polymerase, by Roche (1 mentions)
24 protocols using creatine phosphate
1
Cell Permeabilization and Nuclear Transport
2
In Vitro Drosophila Protein Translation
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As previously done17 (link), whole bodies of flies were homogenized by a plastic pestle in 10 mM HEPES (pH 7.4), 5 mM DTT, and 1x complete protease inhibitor cocktail (Roche) and centrifuged at 14,000 g at 4 °C for 15 min. 0.15 U/µl Micrococcal nuclease (New England BioLabs) and 1 mM CaCl2 were added to the supernatant and incubated at 20 °C for 4 min, followed by the addition of 2 mM EGTA. Translation reaction mixtures contained 40% (v/v) extract, 2 nM luciferase RNA, 100 µM amino acid mix (Promega), 50 mM potassium acetate, 2.5 mM magnesium acetate, 0.1 mM spermidine, 20 U of RNase inhibitor (Fisher), 20 mM creatine phosphate (Roche Applied Science), and 80 ng/µl creatine kinase (Roche Applied Science). The mixture was incubated at 26 °C in a SpectraMax iD3 plate with luminescence measured every 5 min. The slope of the linear part of the luciferase activity curve was used to calculate the rate of in vitro protein translation.
3
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).
4
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)).
5
Ribosome Initiation Complex Purification
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In vitro translation extracts were made from human 293T cells as described23 (link). Lysates were nuclease-treated with 18 gel U/μl micrococcal nuclease (NEB M0247S) in the presence of 0.7 mM CaCl2 for 10 min at 25 °C, and the digestion was stopped by addition of 2.24 mM EGTA. Each translation reaction contained 50% in vitro translation lysate (from 293T cells) and buffer to make the final reaction 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U/ml creatine phosphokinase (Roche), 10 mM HEPES-KOH, pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U/ml murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc. For 48S ribosome subunit initiation complex purification from in vitro translation reactions, reactions were incubated in the presence of GMP-PNP for 20 min at 30 °C and centrifuged for 6 min at 12 000 × g at 4 °C. Lysates were purified by size-exclusion chromatography through a 1 ml column packed with Sephacryl S-400 gel filtration resin (GE Healthcare) and the eluant centrifuged through a 10–25% (w/v) sucrose gradient by centrifugation for 5 h at 36 000 rpm at 4 °C in a Beckman SW40 Ti rotor. Fractions were collected from the gradient and RNA purified by phenol–chloroform extraction and ethanol precipitation, and protein precipitated with trichloroacetic acid.
6
In vitro Translation Assay with S. cerevisiae S30 Extract
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For in vitro translation, an S30 extract from S. cerevisiae was prepared (see Supplemental Experimental Procedures for details) (Hofbauer et al., 1982 (link)). For in vitro translation, 2.5 μl creatine phosphokinase (10 mg/ml; Roche), 7.5 μl CaCl2 (20 mM), 25 μl 10 × translation cocktail (100 mM HEPES/KOH [pH 7.5], 10 mM Mg[OAc]2, 760 mM KCl, 4 mM GTP, 10 mM ATP, 19 amino acid mix [500 μM each; methionine excluded]), 5 μl creatine phosphate (0.6 M; Roche), and 2.5 μl Mg(OAc)2 (100 mM) were mixed with 150 μl S30 and 16 μl 35S-methionine (1,000 Ci/mmol, 10 mCi/ml). The resulting translation mix was aliquoted into 12 μl portions and filled up to 15 μl with sterile H2O, synthetic 18-mer RNA (10–200 pmol) or cycloheximide (7.5 μg/ μl), incubated at 23°C for 30 min, and the products were separated by SDS-PAGE. To test whether translation initiation or elongation are inhibited by the 18-mer RNA, complete reactions, but lacking 35S-methionine and the 18-mer RNA, were assembled and preincubated for 10 min at 23°C. Subsequently the initiated samples were cooled down to 0°C on ice for 5 min followed by the addition of 35S-methionine and 13 μM of the 18-mer RNA. Translation elongation was then carried out for 30 min at 0°C, stopped by TCA precipitation, and the products were quantified by liquid scintillation counting (see Supplemental Experimental Procedures for details).
7
Nuclear Export Assay of NP Mutants
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HeLa cells (1×105) were cultured on coverslips in 12-well plates, transfected with pCAGGS harboring wild-type NP or mutant NP-NES3 Φ1, Φ2, Φ3, or Φ4, and then examined in a nuclear export assay [5] (link). Briefly, the cell membrane was selectively permeabilized by treatment with 50 µg/ml digitonin (Sigma) for 5 min on ice. The cells were then incubated with apyrase (10 units/ml; Sigma) on ice for 5 min to deplete intracellular ATP, washed, and then incubated in PBS to remove all cytoplasmic components. Fresh HeLa cell lysates (prepared by lysing cells in 10 mM Tris-Cl (pH 7.4), 2 mM MgCl2, 3 mM CaCl2, and 0.3 M sucrose) supplemented with protease inhibitor cocktail, 5 mg/ml bovine serum albumin, 1 mM ATP (Sigma), 5 mM creatine phosphate (Roche), and 32 units/ml creatine kinase (Sigma) were added to the cells and incubated at 30°C for 1 h to allow nuclear export to proceed. The cells were then stained with an anti-NP Ab followed by anti-mouse Alexa Fluor 488 and Hoechst 33342. Fluorescence images were taken under a confocal laser-scanning microscope (FV 1000, Olympus).
8
In Vitro Translation Assay from HeLa Cells
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In vitro translation extracts were prepared from cultured HeLa cells37 (link) (ATCC) maintained at 37 °C, 5% CO2 in DMEM supplemented with 10% FBS and 1% non-essential amino acids. To prepare extracts, adherent cells were trypsinized, centrifuged, and washed in PBS. Cell pellets were resuspended in RNAse-free hypotonic buffer containing 10 mM HEPES-KOH (pH 7.6), 10 mM potassium acetate, 0.5 mM magnesium acetate, 5 mM DTT, and EDTA-free protease inhibitor cocktail (Roche). Cell pellets were incubated on ice for 20 min, mechanically disrupted by a 27G syringe, incubated for another 20 min on ice, and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was removed, then brought to 4 μg μL−1 in additional hypotonic buffer. For in vitro translation reactions, lysates were supplemented to final concentrations of 20 mM HEPES-KOH (pH 7.6), 44 mM potassium acetate, 2.2 mM magnesium acetate, 2 mM DTT, 20 mM creatine phosphate (Roche), 0.1 µg µl−1 creatine kinase (Roche), 0.1 mM spermidine, and on average 0.1 mM of each amino acid (with relative amounts approximating those in eukaryotes69 (link)). To this, in vitro transcribed reporter RNAs were added to 4 nM. After incubation at 30 °C for 30 min, luciferase assays were carried out as with RRL reactions.
9
Reconstitution of cis-SNARE Complex
10
Chromatin Isolation from Egg Extracts
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Freshly prepared egg extracts were supplemented with 0.005 mg/ml creatine kinase (Roche, 10127566001), 0.375 mM creatine phosphate (Roche, 10621714001), 0.05 mM ATP (Roche, 10519979001), 0.005 mM EGTA, 1 mM MgCl2. In some experiments, delta-geminin was added (final concentration 1.5 μg/ml) to egg extract to inhibit DNA replication. Permeabilized sperm cells were added to a final concentration of 1000 nuclei/μl of extract and incubated at RT for 2 h with gentle tapping every 10 min. The reaction was stopped by adding 10 volumes of ice-cold Egg Lysis Buffer—Chromatin Isolation Buffer (ELB–CIB, 10 mM Hepes pH 7.8, 250 mM sucrose, 2.5 mM MgCl2, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 1 mM spermidine, 1 mM spermine, 0.1% Triton X-100, 10 mM sodium butyrate, 1× EDTA-free PI Cocktail (Roche, 05056489001)) following Wang et al.42 (link), with slight modifications. Chromatin was isolated via centrifugation at 4000 rpm for 5 min through a 0.3 mL sucrose cushion of ELB–CIB with 0.5 M sucrose underlayered in the tube. The pellet was washed once with ELB–CIB plus 250 mM KCl. Samples were aliquoted (0.5–1 million of cells per tube) and flash frozen in liquid nitrogen.
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