A. baumannii ATCC17978 cells in the exponential phase of growth were treated for 30 min at 37°C with 64 μg/ml OA dissolved in ethanol. Microarray analysis was performed as previously described [30 (link)]. RNA was isolated using the RNeasy Mini kit (Qiagen), and RNA concentrations were estimated by measuring absorbance at 260 nm. The cDNA probes for microarray analysis were prepared by reverse transcription of total RNA (50 μg). The cDNA probes were cleaned up using a Microcon YM-30 column (Millipore) and then coupled to Cy3 or Cy5 (GE Healthcare). The dried Cy3- or Cy5-labeled cDNA probes were then resuspended in 55μl of 2x HI-RPM hybridization buffer (Agilent) containing 30% formamide (v/v), 5× saline-sodium citrate, 0.1% SDS (w/v), and 0.1 mg/ml salmon sperm DNA. The Cy3- or Cy5-labeled cDNA probes were mixed together and hybridized onto a microarray chip (MYcroarray.com). The hybridization images on the slides were scanned using an Axon 4000B (Axon Instruments), and data quantification was performed using GenePix Pro 6.0 (Axon Instrument). Genes that were upregulated more than 1.5-fold in at least two replicates were selected. The microarray data were deposited in the National Center for Biotechnology Information GEO site (under accession number GSE 60239). Microarray data were confirmed by quantitative reverse transcription PCR (RT-PCR).
Microcon ym 30 column
Manufactured by Merck Group
The Microcon YM-30 column is a centrifugal filter device used for the concentration and desalting of protein samples. The column has a molecular weight cutoff of 30 kDa, allowing the retention of proteins above this size while permitting the passage of smaller molecules and salts.
Automatically generated - may contain errors
3 protocols using microcon ym 30 column
1
Oleic Acid Treatment in A. baumannii
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A. baumannii ATCC17978 cells in the exponential phase of growth were treated for 30 min at 37°C with 64 μg/ml OA dissolved in ethanol. Microarray analysis was performed as previously described [30 (link)]. RNA was isolated using the RNeasy Mini kit (Qiagen), and RNA concentrations were estimated by measuring absorbance at 260 nm. The cDNA probes for microarray analysis were prepared by reverse transcription of total RNA (50 μg). The cDNA probes were cleaned up using a Microcon YM-30 column (Millipore) and then coupled to Cy3 or Cy5 (GE Healthcare). The dried Cy3- or Cy5-labeled cDNA probes were then resuspended in 55μl of 2x HI-RPM hybridization buffer (Agilent) containing 30% formamide (v/v), 5× saline-sodium citrate, 0.1% SDS (w/v), and 0.1 mg/ml salmon sperm DNA. The Cy3- or Cy5-labeled cDNA probes were mixed together and hybridized onto a microarray chip (MYcroarray.com). The hybridization images on the slides were scanned using an Axon 4000B (Axon Instruments), and data quantification was performed using GenePix Pro 6.0 (Axon Instrument). Genes that were upregulated more than 1.5-fold in at least two replicates were selected. The microarray data were deposited in the National Center for Biotechnology Information GEO site (under accession number GSE 60239). Microarray data were confirmed by quantitative reverse transcription PCR (RT-PCR).
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A. baumannii ATCC17978 cells in the exponential phase of growth were treated for 30 min at 37°C with 64 μg/ml OA dissolved in ethanol. Microarray analysis was performed as previously described [30 (link)]. RNA was isolated using the RNeasy Mini kit (Qiagen), and RNA concentrations were estimated by measuring absorbance at 260 nm. The cDNA probes for microarray analysis were prepared by reverse transcription of total RNA (50 μg). The cDNA probes were cleaned up using a Microcon YM-30 column (Millipore) and then coupled to Cy3 or Cy5 (GE Healthcare). The dried Cy3- or Cy5-labeled cDNA probes were then resuspended in 55μl of 2x HI-RPM hybridization buffer (Agilent) containing 30% formamide (v/v), 5× saline-sodium citrate, 0.1% SDS (w/v), and 0.1 mg/ml salmon sperm DNA. The Cy3- or Cy5-labeled cDNA probes were mixed together and hybridized onto a microarray chip (MYcroarray.com). The hybridization images on the slides were scanned using an Axon 4000B (Axon Instruments), and data quantification was performed using GenePix Pro 6.0 (Axon Instrument). Genes that were upregulated more than 1.5-fold in at least two replicates were selected. The microarray data were deposited in the National Center for Biotechnology Information GEO site (under accession number GSE 60239). Microarray data were confirmed by quantitative reverse transcription PCR (RT-PCR).
2
Tomato Microarray Hybridization Protocol
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Preparation of the RNA and cDNA, and hybridization reactions were conducted according to
Rodriguez-Saona et al. (2010) (link)
. Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). Enrichment and purification of messenger RNA (mRNA) was conducted with Dynabeads Oligo (dT) 25 (Dynal Biotech, Lake Success, NY). The mRNA was reverse transcribed and labeled with Cy3- and Cy5-deoxyuridine triphosphate (Amersham Biosciences, Piscataway, NJ). The cDNA was purified using a Microcon YM30 column (Millipore, Billerica, MA). Tomato microarrays (TOM1, The Boyce Thompson Institute at Cornell University, Ithaca, NY) were hybridized at 65°C for 8 h. All treatments were dye reversed with Cy3 and Cy5 for a total of six microarray chips using a loop design for four biological replications for each treatment. The complete meta-grid size was 13,440 spots per array. Scanning was conducted with the Packard BioScience Scan Array 3000 equipped with red and green lasers at 10 µm resolution. Spot intensity was quantified using Imagene 5.1 (BioDiscovery, Inc., Marina Del Rey, CA). Gene identifications are available from the Sol Genomics Network (
http://solgenomics.net/ ).
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Preparation of the RNA and cDNA, and hybridization reactions were conducted according to
Rodriguez-Saona et al. (2010) (link)
. Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). Enrichment and purification of messenger RNA (mRNA) was conducted with Dynabeads Oligo (dT) 25 (Dynal Biotech, Lake Success, NY). The mRNA was reverse transcribed and labeled with Cy3- and Cy5-deoxyuridine triphosphate (Amersham Biosciences, Piscataway, NJ). The cDNA was purified using a Microcon YM30 column (Millipore, Billerica, MA). Tomato microarrays (TOM1, The Boyce Thompson Institute at Cornell University, Ithaca, NY) were hybridized at 65°C for 8 h. All treatments were dye reversed with Cy3 and Cy5 for a total of six microarray chips using a loop design for four biological replications for each treatment. The complete meta-grid size was 13,440 spots per array. Scanning was conducted with the Packard BioScience Scan Array 3000 equipped with red and green lasers at 10 µm resolution. Spot intensity was quantified using Imagene 5.1 (BioDiscovery, Inc., Marina Del Rey, CA). Gene identifications are available from the Sol Genomics Network (
3
Purification of Gal/GalNAc Lectin from Trophozoites
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Trophozoites (1.5×107) were lysed in octyl-beta-d-glucopyranoside (30 mM in water), and D-galactose-coated agarose beads (Thermo Scientific-Pierce Protein Biology Products) were used to bind the Gal/GalNAc lectin in the lysate. After several washes in PBS, the lectin was eluted from the beads using 0.1 M galactose in PBS. The proteins in the eluted fractions were concentrated by ultrafiltration on a Microcon YM-30 column (Millipore) and analyzed by Coomassie blue straining and western blotting using antibody against the heavy subunit of Gal/GalNAc lectin in order to confirm the presence of this lectin in the eluent (data not shown).
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