The lipid compositions of bacterial strains were determined following labeling with [1-14C]acetate (Amersham Biosciences). Cultures (1 ml) of Burkholderia s.l. strains were inoculated from precultures grown in the same medium. After addition of 1 μCi of [14C]acetate (60 mCi mmol-1) to each culture, the cultures were incubated overnight. Cells were harvested by centrifugation, washed with 500 μl water once, resuspended in 100 μl water, and then lipids were extracted according to Bligh and Dyer (Bligh and Dyer, 1959 (link)). Aliquots of the lipid extracts were spotted on high performance TLC silica gel 60 plates (Merck, Poole, UK) and separated in two dimensions using chloroform/methanol/water (16:4:1, v/v/v) as a mobile phase for the first dimension and chloroform/methanol/acetic acid (15:3:2, v/v/v) as a mobile phase for the second dimension (Tahara and Fujiyoshi, 1994 (link)). To visualize membrane lipids, developed two-dimensional TLC plates were exposed to autoradiography film (Kodak) or to a PhosphorImager screen (Amersham Biosciences). The individual lipids were quantified using ImageQuant software (Amersham Biosciences) (Vences-Guzmán et al., 2011 (link)).
Phosphorimager screen
Manufactured by Cytiva
Sourced in United Kingdom
The PhosphorImager screen is a lab equipment product used for the detection and quantification of radioactive samples. It functions by capturing and storing the emitted radiation from labeled samples, which can then be analyzed and interpreted by the user.
Automatically generated - may contain errors
Lab products found in correlation
Autoradiography film, by Kodak (2 mentions)
1 14c acetate, by Cytiva (2 mentions)
High performance tlc silica gel 60 plates, by Merck Group (2 mentions)
Imagequant software, by Cytiva (2 mentions)
Hybond nx membrane, by GE Healthcare (2 mentions)
Megaprime dna labeling system, by GE Healthcare (1 mentions)
Typhoon fla 7000, by GE Healthcare (1 mentions)
Lna oligonucleotide probes, by Qiagen (1 mentions)
Semi dry blotting, by Bio-Rad (1 mentions)
Hybond n nylon membrane, by Cytiva (1 mentions)
Neblot kit, by New England Biolabs (1 mentions)
Typhoon 9210 scanner, by Cytiva (1 mentions)
Typhoon 9400 imager, by Cytiva (1 mentions)
Prime it 2 random primer labelling kit, by Agilent Technologies (1 mentions)
Gelred, by Biotium (1 mentions)
Typhoon imager, by GE Healthcare (1 mentions)
Microspin g 25 column, by GE Healthcare (1 mentions)
T4 polynucleotide kinase, by Promega (1 mentions)
Decalabel dna labelling kit, by Thermo Fisher Scientific (1 mentions)
35s sulfate, by Cytiva (1 mentions)
14 protocols using phosphorimager screen
1
Bacterial Lipid Composition Analysis
2
TbGPI2 Expression Analysis by Northern and Southern Blotting
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TbGPI2 mRNA expression was assayed by Northern blotting as described before (44 (link)). Genomic DNA was isolated and digested with BglII or ClaI followed by Southern blotting as previously described (44 (link)). A radioactively labeled probe corresponding to the coding region of TbGPI2 was generated using a Megaprime DNA-labeling system (GE Healthcare) according to the manufacturer’s instructions. Signals were detected by exposure of the blots to a Phosphorimager screen (Amersham Biosciences) followed by scanning with a Typhoon FLA 7000 (GE Healthcare).
3
Small RNA Northern Blot Analysis
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For small RNA northern blot analyses (24 (link)), 4 μg of total RNA or samples of gel-filtration were separated on denaturing 12% polyacrylamide gels containing 8 M urea and transferred to Hybond-NX membrane (GE Healthcare) with semi-dry blotting (Bio-Rad). Membranes were chemically cross-linked (25 (link)) and probed with radiolabeled locked nucleic acid (LNA) oligonucleotide probes (Exiqon, Vedbaek, Denmark) or DNA probes, complementary to the mature miRNAs, as described previously (26 (link)). Signal was detected using X-ray film or Phosphorimager screen (Amersham). Loading rates of miR390 were calculated from the summarized volume intensity of the strongest four signals of fractions representing HMW RISC-bound and -unbound miR390 using ImageLab 1.1 (Bio-Rad).
4
Genomic DNA Extraction and Southern Blot
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Genomic DNA was isolated from chickpea leaves by CTAB method [38 ]. DNA (10 μg) was digested with Bgl II, Hind III, Sal I, Rsa I and Sty I, respectively for CarF-box_PP2 and Bgl II, Sal I and Nco I, respectively for CarF-box_LysM. Digested DNA was separated on 0.8% (w/v) agarose gel, and transferred onto Hybond N nylon membrane (Amersham Biosciences, UK) in 20x SSC. Full length cDNA of CarF-box_PP2 and CarF-box_LysM were used as probe to hybridize the respective blots. The probes were labelled with α-32P-dCTP using NEBlot Kit (NEB, USA). Pre-hybridization was performed at 60°C for 2 h in pre-hybridization buffer (0.1 M sodium phosphate buffer- pH-7.2, 10% SDS and 0.5 M EDTA), followed by hybridization with the labelled probe kept at the same temperature overnight. After hybridization, the blots were washed with 2X SSC, 0.1% SDS for 10 m at 60°C followed by washing with 1X SSC, 0.1% SDS for 10 m at room temperature [39 ]. The membranes were exposed to PhosphorImager screen (Amersham Biosciences, UK) for 24 h and the images were acquired by scanning the screen with Typhoon 9210 scanner (Amersham Biosciences, UK).
5
Resolving S. cerevisiae Chromosomes and Detecting rDNA
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Chromosomes were resolved and rDNA detected as described (El Hage & Housley, 2013 (link)), with the following alterations. Cells were collected from asynchronously growing cultures in mid-log phase without condensin depletion. To resolve S. cerevisiae chromosomes, the gel was run with a 300–900 s switch time, 120° angle, 3 V/cm at 14°C for 68 h. The gel was stained with GelRed (Biotium) in TBE for 1 h, washed twice in 2× TBE for 15 min, and imaged. The gel was transferred onto an N+ Hybond nitrocellulose membrane (GE Healthcare) through capillary transfer as described (El Hage & Housley, 2013 (link)). The rDNA probe for Southern blotting was amplified from the non-transcribed spacer region two (NTS2). The probe was labelled with [α-33P] dATP (3,000 Ci/mmol; Hartmann) using the Prime-It II Random Primer Labelling Kit (Agilent). The probe was then added to the membrane pre-hybridised in QuickHyb Hybridization solution (Agilent), and incubated at 68°C overnight. Membranes were washed twice in 2× SSC, 0.1% SDS for 15 min at room temperature, and twice in 0.5× SSC, 0.1% SDS, rinsed in 50 mM Tris–HCl, pH 7.5 and exposed overnight using a PhosphorImager screen (Amersham Biosciences), and scanned on a Typhoon 9400 Imager.
6
Purification and EMSA of MParD2 Protein
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His6-tagged MParD2 protein was purified as described above and dialysed against 1X EMSA binding buffer (10 mM Tris-HCl pH 8.0, 1 mM DTT, 2.5 mM MgCl2, 40 mM KCl, 0.5 μg ul-1 dI:dC, 5% glycerol). The 200 bp upstream of the parD2 start site, P200parD2 was PCR amplified, end-labeled with [γ-32P]-ATP using T4 polynucleotide kinase (Promega) and purified using G-25 MicroSpin column (GE Healthcare). The MParD2 or the variant proteins were incubated with 20,000 cpm radiolabelled P200parD2 in 1X EMSA binding buffer at room temperature for 20 min, in a reaction mix of 25 μl. Non-denaturing DNA loading dye was added to the reaction mixture and electrophoresed on 5% Native-PAGE gel in 0.5X TBE buffer at 4°C at 60 V till bromophenol blue reached the bottom. The gel was dried and exposed to a phosphorimager screen (Amersham Biosciences) overnight and scanned in a Typhoon Imager (GE Healthcare).
7
Northern Blot Analysis of p14 Expression
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To detect p14 6 μg of same RNA as was used to detect miR168 was separated on formaldehyde agarose gels, blotted onto Hybond NX membrane (GE Healthcare) with capillary transfer, UV crosslinked and subjected to hybridization with radiolabeled PCR product of p14 at 65ºC as described previously (39 (link)). Probe was labelled using DecaLabel DNA labelling Kit of Thermo Fisher Scientific according to the manufacturer's instructions. Image was created with phosphorimager screen (Amersham).
8
Bacterial Lipid Composition Analysis
9
Nuclear Factor-κB Activation Assay
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RAW264.7 macrophages cells were plated in 6-well plates (1×106 cells/well). The cells were either pretreated with 5 μg/mL AA for 30 minutes or with complete medium. The cells were then either stimulated with 1 μg/mL LPS for 30 minutes, or they were left UT. Nuclear extracts were prepared and frozen at −70°C until use.
The EMSA protocol conducted was as previously described23 (link) with some minor modifications. Also, 10 μg of the nuclear extract proteins were mixed with 12 μL of a mixture containing binding buffer, poly-deoxy-inosinic-deoxy-cyti-dylic acid (Poly[d(I-C)]), bovine serum albumin (BSA), and an NFκB-specific 32P-labeled oligonucleotide comprising the sequence 5′-AGCTATGTGGGTTTTCCC
The EMSA protocol conducted was as previously described23 (link) with some minor modifications. Also, 10 μg of the nuclear extract proteins were mixed with 12 μL of a mixture containing binding buffer, poly-deoxy-inosinic-deoxy-cyti-dylic acid (Poly[d(I-C)]), bovine serum albumin (BSA), and an NFκB-specific 32P-labeled oligonucleotide comprising the sequence 5′-AGCTATGT
10
Mapping 2'-O-Methylation Sites
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A subset of 2′-O-Me sites with low (RMS < 0.75) or inconsistent scores and sites without a plausible SNORD were further assessed by the high/low dNTP-concentration primer extension method (Maden 2001 (link)). Primers were designed based on the predicted 2′-O-Me sites and a list of all the primers can be found in Supplemental Table S5 . Reverse transcription of 1 µg of whole-cell RNA from appropriate developmental stages were performed in 20 µL 1× RT buffer at 42°C for 60 min supplemented with 1 µL AMV RT (Promega, 20 U) at low and high dNTP concentrations (0.01 mM and 1 mM, respectively). The cDNA generated were separated on an 8% UPAG together with a sequencing ladder. Dried gels were exposed to Phosphor Imager Screens and scanned using a Typhoon Biomolecular Imager (Amersham) to visualize the radioactive signals from the probes. Images were analyzed using Fiji software.
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