Whole cell extracts were prepared with the cell lysis buffer (10 mM Tris-HCl, pH 7.4, 1% SDS, and 1 mM Na3VO4) as described in our previous studies [57 (link), 58 (link)]. Protein extracts were subjected to Western Blot with the indicated primary antibodies, and probed with the AP-conjugated secondary antibody together with the enhanced chemifluorescence system as described in a previous reports [57 (link), 58 (link)]. The images were acquired by scanning with the PhosphorImager Typhoon FLA 7000 (GE, Pittsburgh, PA).
Typhoon fla 7000 phosphorimager
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Sweden
The Typhoon FLA 7000 is a phosphorimager designed for the detection and analysis of radiolabeled samples. It utilizes a laser-based scanning system to capture high-resolution images of phosphor-based detection methods, such as autoradiography and phosphor screens. The instrument is capable of digitizing and quantifying the signals from these samples.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant tl, by GE Healthcare (4 mentions)
Imagequant tl software, by GE Healthcare (4 mentions)
Genomic dna handbook, by Qiagen (2 mentions)
Rnase 3, by Thermo Fisher Scientific (2 mentions)
Sybr green 2, by Biozym (2 mentions)
Low range ssrna ladder, by New England Biolabs (2 mentions)
Amersham hyperfilm mp, by GE Healthcare (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Expresshyb hybridization solution, by Takara Bio (2 mentions)
Hybond xl membrane, by Cytiva (2 mentions)
G25 microspin columns, by GE Healthcare (2 mentions)
Zeta probe membrane, by Bio-Rad (2 mentions)
Gotaq green, by Promega (2 mentions)
Proteinase k, by Roche (2 mentions)
Multi gauge software, by Fujifilm (2 mentions)
Phosphorimaging screen, by GE Healthcare (2 mentions)
Imagequant tl v8, by GE Healthcare (2 mentions)
Halt proteinase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Easytag express35s protein labeling mix, by PerkinElmer (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
67 protocols using typhoon fla 7000 phosphorimager
1
Western Blot Analysis of Whole Cell Extracts
2
Radioactive Enzymatic Assays for Lipid Biosynthesis
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Enzymatic assays monitored either 1) the hGPT-mediated transfer of [14C]phospho-GlcNAc from [14C]UDP-GlcNAc to dolichyl phosphate (C55-Dol-P), forming [14C]Dol-PP-GlcNAc, or 2) the MraYAA-mediated transfer of [14C]phospho-MurNAc-pentapeptide from [14C]UDP-MurNAc-pentapeptide(DAP) ([14C]UM5A) to undecaprenyl phosphate (C55-P), forming [14C]Lipid I. A thin-layer chromatography (TLC)-based radiochemical assay 51 (link) was used, which was optimized for hGPT and MraYAA-catalyzed reactions for the experiments described below. All reactions were quenched by spotting a 2-μL aliquot on a silica gel 60 thin layer chromatography (TLC) plate (EMD Millipore). The products and substrates were separated by TLC using isopropanol/ammonium hydroxide/water (6:3:1; v/v/v) as the mobile phase. The spots corresponding to product and substrate were visualized via Phosphorimager (Typhoon FLA 7000, GE Healthcare LifeSciences). The spot intensity was quantified using the ImageQuant TL (GE Healthcare LifeSciences). All experiments were performed in triplicate.
3
Quantitative Western Blot Analysis
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Whole-cell extracts were prepared with the cell lysis buffer (10 mM Tris-HCl, pH 7.4, 1% SDS, and 1 mM Na3VO4) as described in our previous studies [54 (link)]. Protein extracts were subjected to western blot with the indicated primary antibodies, and probed with the AP-conjugated secondary antibody together with the enhanced chemifluorescence system as described in a previous report [54 (link)]. The images were acquired by scanning with the PhosphorImager Typhoon FLA 7000 (GE, Pittsburgh, PA). The densitometry analyses of the specific protein degradation were performed using software Image J (NIH, Bethesda, MD) to obtain the protein half-life, which is calculated using the formula described in a published article [55 (link)].
4
Radioactive Enzymatic Assays for Lipid Biosynthesis
5
Genomic DNA Extraction and Southern Blotting
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Total genomic DNA was extracted according to the protocol of the QIAGEN Genomic DNA Handbook, using genomic-tip 100/G columns (a detailed protocol is provide in SI Materials and Methods ). Two-dimensional gels were prepared and run as previously described (74 (link)). The DNA samples were digested by the enzyme indicated in the figures and then transferred to a Nylon Gene Screen Plus membrane (NEN) for Southern blotting analysis with specific probes against the loci. Primers used for amplification of the probes are available upon request. Signals were detected using a PhosphorImager Typhoon FLA 7000 (GE Healthcare) and quantified as previously described (75 (link)).
6
Genomic DNA Extraction and 2D Gel Electrophoresis
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Total genomic DNA was extracted according to the protocol of the QIAGEN Genomic DNA Handbook, using genomic-tip 100/G columns. 2D gel electrophoresis was carried out as originally described by Brewer and Fangman [78] (link). The DNA samples were digested with HindIII or SacI/ApaL1, for ARS305 and ARS607 detection respectively, and then blotted onto a Nylon Gene Screen Plus membrane (NEN). Membranes were probed with the BamHI-NcoI 3.0 kb fragment which spans ARS305 and was purified from plasmid A6C-110 (kindly provided by C. Newlon, uMDNJ, Newark, NJ), or probed with a 3.0 kb PCR product that spans ARS607. Signals were detected using a PhosphorImager Typhoon FLA 7000 (GE Healthcare).
7
Measuring p63α Protein Synthesis
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UROtsa(Nonsense) and UROtsa(shXIAP) were incubated with methionine–cysteine-free DMEM (Gibco-BRL, Grand Island, NY) containing 2% dialyzed fetal calf serum (Gibco-BRL, Grand Island, NY) in presence of MG132 (50 μM) for 1 hours. The cells were then incubated with 2% fetal bovine serum methionine–cysteine-free DMEM containing 35S-labeled methionine/cysteine (250 mCi per dish, Trans 35S-label; Perkin Elmer, Boston, MA) for the indicated time periods. The cells were extracted with lysis buffer (Cell Signaling, Beverly, MA) containing complete protein inhibitor mixture (Roche, Branchburg, NJ) on ice for 10 mins. Total lysate of 500 mg was incubated with anti-p63α antibody-conjugated agarose beads (R&D Systems, Minneapolis, MN) at 4°C overnight. The immunoprecipitated samples were washed with the cell lysis buffer five times, heated at 100°C for 5 min and then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The 35S-labeled p63α protein was detected with the PhosphorImager Typhoon FLA 7000 (GE, Pittsburgh, PA).
8
RNase III Cleavage Assay for RNA
9
RNase III Cleavage Assay for RNA
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In vitro-transcribed and purified RNAs were boiled in a thermocycler at 95°C for 10 min, cooled down to room temperature by sitting on a bench for 10 min, and kept on ice. Cleavage reactions were prepared by adding 40 ng of RNA; 1, 0.2, 0.04, 0.008, or 0 units of RNase III (Invitrogen); and water in the supplemented buffer to a total volume of 10 μL. After incubation for 5 min at 37°C, the reaction was stopped by adding an RNA loading buffer (0.025% bromophenol blue, 0.025% SDS, 0.025% xylene cyanol, 18 mM EDTA (pH 8.0), 93.64% formamide) on ice. The mixture then was boiled in a thermocycler at 95°C for 10 min, resolved on an 8% polyacrylamide gel (20 × 20 cm) containing 7 M urea at 300 V for 210 min, stained with SYBR Green II (Biozym), and visualized on a Phosphorimager (Typhoon FLA 7000, GE Healthcare). The Low Range ssRNA Ladder (New England Biolabs) was used as a marker. For the assays with the leader-repeat-spacer RNA for LrhCas9, the RNA was truncated within the leader and the spacer to avoid cleavage of irrelevant secondary structures formed internally within either domain.
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In vitro-transcribed and purified RNAs were boiled in a thermocycler at 95°C for 10 min, cooled down to room temperature by sitting on a bench for 10 min, and kept on ice. Cleavage reactions were prepared by adding 40 ng of RNA; 1, 0.2, 0.04, 0.008, or 0 units of RNase III (Invitrogen); and water in the supplemented buffer to a total volume of 10 μL. After incubation for 5 min at 37°C, the reaction was stopped by adding an RNA loading buffer (0.025% bromophenol blue, 0.025% SDS, 0.025% xylene cyanol, 18 mM EDTA (pH 8.0), 93.64% formamide) on ice. The mixture then was boiled in a thermocycler at 95°C for 10 min, resolved on an 8% polyacrylamide gel (20 × 20 cm) containing 7 M urea at 300 V for 210 min, stained with SYBR Green II (Biozym), and visualized on a Phosphorimager (Typhoon FLA 7000, GE Healthcare). The Low Range ssRNA Ladder (New England Biolabs) was used as a marker. For the assays with the leader-repeat-spacer RNA for LrhCas9, the RNA was truncated within the leader and the spacer to avoid cleavage of irrelevant secondary structures formed internally within either domain.
10
Quantification of Cas10-Csm crRNA Species
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Total crRNAs were extracted from purified Cas10-Csm complexes using the TRIzol Reagent (Invitrogen, NY, USA) according to Hatoum-Aslan et al. (2014) (link). Extracted crRNAs were phosphorylated with T4 Polynucleotide Kinase (New England Biolabs, MA, USA), radiolabeled with γ-[32P]-ATP (PerkinElmer, MA, USA), and resolved on a 15% Urea PAGE gel. The gel was exposed for 10 min to a storage phosphor screen and visualized using a Typhoon FLA 7000 phosphor imager (GE Healthcare Bio-Sciences, PA, USA). For densitometric analysis, the ImageQuant software was used. Percent of intermediate crRNAs was determined with the following equation: ((intensity of intermediate crRNA signal (71 nt) ÷ sum of signal intensities for the dominant crRNA species (71 nt + 43 nt+37 nt+31 nt))×100%). The reported values represent an average of 3–5 replicates (±S.D.), as indicated in the Figure 2 legend and Figure 2—source data 1 .
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