Fluorescent aRNA targets were prepared from 1 µg total RNA samples using OneArray® Amino Allyl aRNA Amplification kit (Phalanx Biotech Group) and Cy5 dye (GE Healthcare Life Sciences). Fluorescent targets were hybridized to the Mouse Whole Genome OneArray® with Phalanx hybridization buffer using Phalanx Hybridization system. After 16 h hybridization, non-specific binding targets were removed using saline and sodium citrate buffer. The slides were scanned using a DNA Microarray Scanner (Model G2505C; Agilent Technologies, Inc.). The Cy5 fluorescent intensities of each spot were analyzed using GenePix 4.1 software (Molecular Devices, LLC, Sunnyvale,. CA, USA). Each single sample was at least assessed twice in terms of technical or biological replicates under the reproducibility >0.975. The signal intensity was loaded into Rosetta Resolver system® (Rosetta Biosoftware, Seattle, WA, USA) for data pre-processing and 75 percentile centering normalization was applied. The errors of the sample were estimated by using the error-weighted approach at the same time. The fold change and P-value for pair-wise sample comparison were calculated for evaluating differentially expressed genes (DEGs). The criteria with log2|fold change|≥0.5 and P<0.05 were used for further analysis.
Cy5 dye
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
Cy5 dye is a fluorescent labeling agent commonly used in various laboratory techniques, such as gene expression analysis, protein detection, and high-throughput screening. It is known for its ability to emit light in the red-orange region of the visible spectrum, making it suitable for specific applications that require detection in that wavelength range.
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Lab products found in correlation
Cyanine3 (cy3), by GE Healthcare (6 mentions)
Cy3 dye, by GE Healthcare (4 mentions)
Genepix 4, by Molecular Devices (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Microarray slides, by Corning (2 mentions)
Dna microarray scanner, by Agilent Technologies (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Jib 04, by Merck Group (1 mentions)
Trizol, by Merck Group (1 mentions)
Jib 04, by Cayman Chemical (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Polyclonal anti gfp, by Takara Bio (1 mentions)
Model g2505c, by Agilent Technologies (1 mentions)
Cydye, by Merck Group (1 mentions)
Sephadex g 25 column, by GE Healthcare (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Carbo free blocking solution, by Vector Laboratories (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Ettan daltsix electrophoresis unit, by GE Healthcare (1 mentions)
Typhoon 9400 imager, by GE Healthcare (1 mentions)
24 protocols using cy5 dye
1
Mouse Whole Genome Expression Analysis
2
Microarray Analysis of Lovastatin-Treated HCP4 Cells
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Total RNA of HCP4 cells with or without Lovastatin treatment was extracted by miRNeasy Mini Kit (Qiagen) and 1 µg or 0.25 µg of total RNAs were used for 3D-Gene mRNA. Microarray analysis was performed using the 3D-Gene mRNA microarray platforms (TORAY, Kamakura, Japan). Briefly, for 3D-Gene mRNA microarray analysis, total RNA was transcriptionally amplified once using Arcturus® Paradise® PLUS 2 Round Kit–Amino Allyl (Life Technologies Corporation, Carlsbad, CA USA) according to the manufacturerʼs protocol. Obtained amino-allyl labeled antisense RNA (10 µg of aRNA) was labeled with Cy5-dye (GE Healthcare, Buckinghamshire, England) according to the manufacturerʼs protocol. The Cy5-labeled aRNA pools and hybridization buffer, and hybridized for 16 h. The hybridization was performed using the supplierʼs protocols (www.3d-gene.com ). Detected signals for each gene were normalized by global normalization method (the median of the detected signal intensity was adjusted to 25.
3
Transcriptomic Analysis of Malaria Gametocytes
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DNA microarrays (60-mer, Agilent Technologies, USA) based on the full P. falciparum genome as previously described [96 (link)] were used to assess global transcriptomic changes in gametocytes treated with JIB-04. Day 2 and day 3 gametocyte cultures (1–3% gametocytaemia, 4% haematocrit) were treated with 5 µM JIB-04 (Cayman Chemicals) for 24 h followed by isolation of gametocytes using 0.01% (w/v) saponin. Total RNA was isolated with a combination of TRIzol (Sigma-Aldrich, USA) and phenol–chloroform extraction and subsequently used to synthesise cDNA as previously described [96 (link)] for the untreated and JIB-04 treated day 2 and day 3 gametocyte samples. Sample cDNA was labelled with Cy5 dye (GE Healthcare, USA) prior to hybridisation to arrays with an equal amount (350–500 ng) of Cy3-labelled (GE Healthcare, USA) reference pool containing equal amounts of cDNA from each gametocyte sample and mixed stage 3D7 asexual parasites. After hybridisation, the slides were scanned on a G2600D (Agilent Technologies, USA) scanner and normalised signal intensities for each oligo were extracted using the GE2_1100_Jul11_no_spikein protocol and Agilent Feature Extractor Software (v 11.5.1.1) as described before [96 (link)].
4
Fluorescent Protein Tagging in MDCK and CHO Cells
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MDCK cells were grown in DMEM (Sigma–Aldrich) containing 5% FBS. MDCK cells were co-transfected with sequences encoding for GFP-FR and mCherry-PLAP (placental alkaline phosphatase) or mCherry-p75 (refer to ref. [5 (link)]). MDCK cells transfected stably with mGFP-FR or transiently with mGFP-uPAR were previously described [5 (link)]. CHO cells were grown in HAM's F12 medium containing 10% FBS and were transfected with different cDNAs: cells stably expressing GFP-FR (kind gift of Dr S. Mayor, NCBS, Bangalore, India) were transiently co-transfected with mCherry-PLAP; CHO cells were transiently transfected with c-DNA encoding for PLAP.
We used the following antibodies: polyclonal anti-GFP (Clontech) and polyclonal anti-PLAP (from Rockland). We generated Fab fragments (using the protocol provided by Pierce) using either GFP or PLAP antibody and then they were coupled to CY5 dye (GE Healthcare Life Science).
We used the following antibodies: polyclonal anti-GFP (Clontech) and polyclonal anti-PLAP (from Rockland). We generated Fab fragments (using the protocol provided by Pierce) using either GFP or PLAP antibody and then they were coupled to CY5 dye (GE Healthcare Life Science).
5
Mouse Whole Genome Expression Profiling
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The Mouse Whole Genome OneArray® v2 (Phalanx Biotech Group, Taiwan) contains 27,307 DNA oligonucleotide probes, and each probe is a 60-mer designed in the sense direction. Among the probes, 26,423 probes correspond to the annotated genes in RefSeq v51 and Ensembl v65 database and the other 884 probes are used for control. The detailed descriptions of the gene array list are available at http://www.phalanx.com.tw/products/MOA.php .
Fluorescent RNA targets were prepared from 1 µg total RNA samples using OneArray® Amino Allyl aRNA Amplification Kit (Phalanx Biotech Group, Taiwan) and Cy5 dye (GE Healthcare). They were then hybridized to the Mouse Whole Genome OneArray® with Phalanx hybridization buffer using Phalanx Hybridization System. After 16 hrs hybridization, non-specific binding targets were washed away. The slides were scanned using a DNA microarray Scanner (Model G2505C, Agilent Technologies). The Cy5 Fluorescent intensities of each spot were analyzed by GenePix 4.1 software (Molecular Devices).
Fluorescent RNA targets were prepared from 1 µg total RNA samples using OneArray® Amino Allyl aRNA Amplification Kit (Phalanx Biotech Group, Taiwan) and Cy5 dye (GE Healthcare). They were then hybridized to the Mouse Whole Genome OneArray® with Phalanx hybridization buffer using Phalanx Hybridization System. After 16 hrs hybridization, non-specific binding targets were washed away. The slides were scanned using a DNA microarray Scanner (Model G2505C, Agilent Technologies). The Cy5 Fluorescent intensities of each spot were analyzed by GenePix 4.1 software (Molecular Devices).
6
Quantitative Proteomic Profiling of 3xTg-AD Mice
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All the samples from WT mice, and 3×Tg-AD mice receiving sham, FUS, or FUS/MB treatment were diluted to a concentration of 5 μg/μL with DIGE-specific lysis buffer. The protein sample (25 μg) was labeled with 200 pM Cy3 (GE Healthcare, USA) or Cy5 dye (GE Healthcare, USA). The internal standard prepared by pooling together equal aliquots of all the samples was labeled with Cy2 (GE Healthcare, USA). The CyDye stock with a final concentration of 1 nM/μL was prepared by reconstitution in 99.8% anhydrous N, N-Dimethylformamide (DMF, Sigma 227056). Protein labeling was performed by incubation with the working solution of each CyDye (200 pM/μL) on ice in the dark for 30 min, and terminated by the addition of 10 mM lysine (Sigma, USA) for 10 min at 4 °C in the dark. Dye swap was performed with each of the sample types in the study such that an equal number were labeled with Cy3 as with Cy5. After labeling, the Cy2-, Cy3-, and Cy5-labeled samples were mixed and then resolved in rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 2.8% DTT, 0.5% IPG buffer (pH 3-11 NL) and 0.002% bromophenol blue) to a final volume of 450 μL prior to transfer onto immobilized pH gradient strips.
7
Lectin Cytochemistry for Cell Imaging
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Lectin cytochemistry was performed as described previously (21 (link), 30 (link)). First of all, 100 μg of Maackia amurensis lectin-I (MAL-I) was labeled with Cy5 dye (GE healthcare) and then purified by a Sephadex-G25 column (GE healthcare). The cells (1 × 105 cells) were inoculated into confocal culture dishes (JingAn Biotechnology Co., Ltd Shanghai China) and cultured in complete medium for 12 h. After that, the cells were washed three times with 10 mM of PBS (5 min each), and immobilized by incubating with 0.2% (v/v) Triton X-100 in 4% (v/v) paraformaldehyde for 30 min at room temperature (RT). Subsequently, the dishes were blocked by Carbo-Free Blocking Solution (Vector labs, Burlingame, CA) for 1 h prior to staining at RT. The cells were then incubated with blocking solution containing 100 μg/mL of Cy5 labeled MAL-I for 4 h with protection from light at RT. After washing for three times with 10 mM of PBS, cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI, 1 μg/mL in 10 mM of PBS) (Roche; Basel, Swatzerland) for 10 min and then washed with 10 mM of PBS again. Finally, the cells were scanned by Laser Scanning Confocal Microscope FV 1000(Olympus, Tokyo, JPN) at the same exposure time and shown on the same scale.
8
Fluorescent Labeling of HEp-2 Proteins
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This step was exclusively used for the FBIP approach. Fifty micrograms per strip of HEp-2 extract protein were labeled with 400 pmol Cy3 dye (GE Healthcare), according the manufacturer’s instructions. Cy5 dye labeling was used when a post-staining step with Deep Purple (DP, GE Healthcare) was performed.
9
2D-DIGE Proteome Analysis Protocol
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2D-DIGE method was conducted following the general procedure described in the study by Otte et al.13 Briefly, 50 μg protein per biological replicate was labeled with 2D-DIGE Cy3 Dye for control and Cy5 Dye for the clinorotated group following the protocol of the manufacturer (GE Healthcare Life Sciences, Munich, Germany). An internal pooled standard was prepared by pooling 25 μg protein of all biological replicates and labeling of 300 μg with 2D-DIGE Cy2 Dye. For each 2D-DIGE gel, 50 μg of one Cy3-labeled control replicate, 50 μg of one Cy5-labeled clinorotated replicate, and 50 μg of Cy2-labeled internal pooled standard were combined.
For first dimension separation, 24-cm gel strips (DryStrips pH 4–7, GE Healthcare) and an IPGPhore (Pharmacia Biotech, Uppsala, Sweden) were used. For second dimension separation, the gel strips were fixed on top of lab cast gels and electrophoresis was performed using an ETTANDaltsix electrophoresis unit (GE Healthcare Life Sciences). During the whole 2D-DIGE procedure, all five biological replicates were processed in parallel.
For first dimension separation, 24-cm gel strips (DryStrips pH 4–7, GE Healthcare) and an IPGPhore (Pharmacia Biotech, Uppsala, Sweden) were used. For second dimension separation, the gel strips were fixed on top of lab cast gels and electrophoresis was performed using an ETTANDaltsix electrophoresis unit (GE Healthcare Life Sciences). During the whole 2D-DIGE procedure, all five biological replicates were processed in parallel.
10
Proteomic Analysis of Rat Serum Samples
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A total of 50 μg of serum samples were labeled with Cy3 or Cy5 dye, according to the manufacturer's protocol (GE Healthcare UK Ltd., Buckinghamshire, UK). An equal amount of sample from eight LEA rats and eight BN rats was pooled, labeled with Cy2 dye, and used as an internal standard. Three labeled protein samples (Cy3, Cy5 and Cy2) were combined per gel, and were subsequently applied to 24‐cm immobilized pH gradient strips, pH 4–7 (GE Healthcare). Isoelectric focusing was carried out using an IPGphor system (GE Healthcare) with a total of 95 kVh. The proteins were then separated on 10–15% gradient acrylamide gel at 1 W per gel. The gels were scanned using a Typhoon 9400 imager (GE Healthcare), and analyzed with DeCyder software (version 6.5; GE Healthcare). Protein spots with an average ratio ≥1.5 or ≤−1.5 and a P‐value <0.05 were considered to be differentially expressed protein spots, and were selected for identification.
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