Streptavidin cy3

Manufactured by Cytiva

Sourced in United States

Streptavidin-Cy3 is a conjugate of the protein streptavidin and the fluorescent dye Cy3. Streptavidin has a high affinity for the small molecule biotin, allowing it to be used in various bioanalytical techniques that involve biotin-labeled molecules. The Cy3 dye provides a fluorescent signal that can be detected and measured.

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Lab products found in correlation

10 protocols using streptavidin cy3

Illumina-Based Gene Expression Profiling in MDA-MB-231 Cells

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For gene expression profiling in MDA-MB-231 cell lines, we used the Illumina HumanHT-12-v4-BeadChip (Illumina). Total RNA isolated from the cell lines with control siRNA or PIN1 siRNA or treated with DMSO or KPT-6566 for 48 h were reverse transcribed and amplified, according to standard protocols and in vitro transcription was then carried out to generate cRNA. cRNA was hybridized onto each array (three biological replicates for the KPT-6566 condition and two for PIN1 siRNA condition) and then labelled with Cy3-streptavidin (Amersham Biosciences). The array was then scanned using a BeadStation 500 system (Illumina). The probe intensities were calculated and normalized using GenomeStudio Data Analysis Software's Gene Expression Module (GSGX) Version 1.9 (Illumina). Further data processing was performed in the R computing environment version 3.2 (http://www.r-project.org/), with BioConductor packages (http://www.bioconductor.org/).

Campaner E., Rustighi A., Zannini A., Cristiani A., Piazza S., Ciani Y., Kalid O., Golan G., Baloglu E., Shacham S., Valsasina B., Cucchi U., Pippione A.C., Lolli M.L., Giabbai B., Storici P., Carloni P., Rossetti G., Benvenuti F., Bello E., D'Incalci M., Cappuzzello E., Rosato A, & Del Sal G. (2017). A covalent PIN1 inhibitor selectively targets cancer cells by a dual mechanism of action. Nature Communications, 8, 15772.

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Illumina Gene and miRNA Profiling

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RNA samples were processed for gene and miRNA profiling on the Illumina platform at the Functional Genomics Facility of INT. For gene profiling analysis, 800 ng of total RNA was reverse transcribed, biotin-labeled, and amplified using Illumina RNA TotalPrep Amplification kit (Ambion, Life Technologies, Grand Island, NY) as per the manufacturer’s instructions. One μg of each cRNA amplified sample was added to Hyb E1 hybridization buffer containing 37.5% (w/w) formamide and hybridized to array HumanHT-12-v3 expression Bead Chip (Illumina, Inc., San Diego, CA) at 58°C for 18 h. Array chips were washed and stained using 1 μg/mL of Cy3-streptavidin (Amersham Biosciences). For miRNA profiling, miRNAs were amplified with the Illumina human_v2 MicroRNA expression profiling kit, based on the DASL (cDNA-mediated Annealing, Selection, Extension, and Ligation) assay according to the manufacturer’s instructions. Briefly, 400 ng/sample of total RNA was converted to cDNA. After annealing with miRNA-specific oligo pools, PCR amplification and fluorescent labeling, probes were hybridized on Illumina miRNA BeadChips, allowing analysis of 1146 sequences covering 97% of miRNAs present in the miRBase v12.0. After hybridization, fluorescent signals were detected by the Illumina BeadArray^TM reader.

Huang X., Dugo M., Callari M., Sandri M., De Cecco L., Valeri B., Carcangiu M.L., Xue J., Bi R., Veneroni S., Daidone M.G., Ménard S., Tagliabue E., Shao Z., Wu J, & Orlandi R. (2015). Molecular portrait of breast cancer in China reveals comprehensive transcriptomic likeness to Caucasian breast cancer and low prevalence of luminal A subtype. Cancer Medicine, 4(7), 1016-1030.

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Illumina HumanHT-12 V4 BeadChip Transcriptome Analysis

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440 ng total RNA was used for in vitro transcription, overnight for 14h using biotin-11-dUTP for labeling of the cRNA product using the Illumina TotalPrep RNA amplification kit (Ambion). The cRNA yields were quantified with a spectrophotometer and the labeled cRNA (500 ng) was hybridized to HumanHT-12 V4 BeadChip™ arrays (Illumina) containing 47,231 transcripts (targeting approximate 31,000 annotated genes) at 55 °C overnight following staining with 1 μg/mL streptavidin-Cy3 (Amersham Biosciences) for visualization. Washing of the arrays was performed using Illumina high-stringency wash buffer for 30 min at 55 °C, followed by scanning according to standard Illumina protocols. Data for probe intensity and detection were captured with Illumina BeadStudio software using background correction and samples were quantile normalized (Schmid, Baum et al. 2010 (link)) and mean centered per chip (Kitchen, Sabine et al. 2010 (link)). Quality standards for hybridization, staining, labeling, and background signal, along with basal level of housekeeping gene expression for each chip were confirmed. The microarray data are deposited in NCBI's Gene Expression Omnibus and are accessible through GEO series accession number GSE63095.

Schisler J.C., Ronnebaum S.M., Madden M., Channell M.M., Campen M.J, & Willis M.S. (2015). Endothelial Inflammatory Transcriptional Responses Induced by Plasma Following Inhalation of Diesel Emissions. Inhalation toxicology, 27(5), 272-280.

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Profiling Gene Expression in Idiopathic Interstitial Pneumonia

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Gene expression profiles of frozen tissue specimens from 12 IIP patients, derived from the central part of the surgical lung biopsy, were analyzed by GP Biosciences Ltd. (Kanagawa, Japan). RNA quality was verified using the RNA6000 Nano Assay on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Illumina Human WG-6 v3 BeadArrays (Illumina Inc., San Diego, CA, USA) with about 48,000 transcripts were used according to the manufacturer’s instructions. An Illumina TotalPrep RNA amplification kit (Ambion, Inc., Austin, TX, USA) was used to obtain biotin-labeled cRNA from 500 ng total RNA. As a control probe, normal human lung poly(A) RNA (BD Biosciences Clontech) was amplified under the same conditions. cRNA was synthesized overnight (18 h), labeled, and hybridized to the chip at 58 °C overnight. Hybridized arrays were labeled with streptavidin-Cy3 (PA43001; Amersham, Buckinghamshire, UK) and scanned with an Illumina BeadArray reader (Illumina Inc.). Scanned images were imported into BeadStudio v3 software (Illumina Inc.) for extraction, quality adjustment, and quantile normalization. Satisfactory quality was observed for all arrays and samples.

Horimasu Y., Ishikawa N., Taniwaki M., Yamaguchi K., Hamai K., Iwamoto H., Ohshimo S., Hamada H., Hattori N., Okada M., Arihiro K., Ohtsuki Y, & Kohno N. (2017). Gene expression profiling of idiopathic interstitial pneumonias (IIPs): identification of potential diagnostic markers and therapeutic targets. BMC Medical Genetics, 18, 88.

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Immunofluorescence Analysis of Plasma Cell Markers

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Immunofluorescence experiments were done as reported before (10 (link)). The technique is based on the ability of biotinylated TG2, followed by labeled streptavidin, to selectively bind TG2-specific antibody deposits inside of plasma cells. Briefly, biopsy specimens stored at -70°C were thawed and rinsed with PBS. Primary reagents were incubated in 1.2% BSA in PBS, for 45 min at RT. Sections were extensively rinsed with PBS, and secondary reagents were incubated in 12% BSA in PBS, for 30 min at RT. Sections were rinsed with PBS and mounted. The following antibodies and reagents were used: mouse anti-CD138 (1:25, AbD Serotec), recombinant human TG2 produced in E. coli and biotinylated as reported before (10 (link)) (5 μg/ml), mouse anti-TG2 mAb CUB7402 (1:1000, Neomarkers), anti-TACI (1:200, sc-7332 N-19), anti-BCMA (1:200, sc-11743 N-16), anti-BAFF-R (1:150, eBioscience 8A7) primary antibodies. Secondary antibodies were used as follows: Alexa Fluor 546/488/649-conjugated anti-goat, anti-mouse and anti-rabbit pAbs (1:1000, LifeTechnologies), streptavidin-cy2 (1:1000, Amersham Biosciences), streptavidin-cy3 (1:2000, Amersham Biosciences), anti-mouse-cy3 (1:1500, Southern Biotech), anti-IgA-FITC (1:40, Dako). DAPI was used to counterstain nuclei.

Di Niro R., Snir O., Kaukinen K., Yaari G., Lundin K.E., Gupta N.T., Kleinstein S.H., Cols M., Cerutti A., Mäki M., Shlomchik M.J, & Sollid L.M. (2015). Responsive population dynamics and wide seeding into the duodenal lamina propria of transglutaminase-2-specific plasma cells in celiac disease. Mucosal Immunology, 9(1), 254-264.

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Illumina Whole Genome Expression Profiling

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Illumina Whole Genome HumanWG6 v4 Expression BeadChips (Illumina Inc, San Diego, CA, USA) were used. Each RNA sample (0.5 μg) was amplified using the Illumina TotalPrep RNA amplification kit with biotin UTP (Enzo Life Sciences, Inc., Farmingdale, NY, USA) labeling. T7 oligo(dT) primers were used to generate single-stranded cDNA followed by a second-strand synthesis to generate double-stranded cDNA, which is then column-purified. In vitro transcription was done to synthesize biotin-labeled cRNA using T7 RNA polymerase. The cRNA was then column-purified and checked for size and yield using the Bio-Rad Experion system (Bio-Rad Laboratories, Hercules, CA, USA). cRNA (1.5 μg) was then hybridized for each array using standard Illumina protocols with streptavidin-Cy3 (Amersham Biosciences, Piscataway, NJ, USA) being used for detection. Slides were scanned on an Illumina Beadstation (Illumina Inc).

Karanam N.K., Srinivasan K., Ding L., Sishc B., Saha D, & Story M.D. (2017). Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines. Cell Death & Disease, 8(3), e2711-.

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High- and Low-LET Radiation-Induced HCC Transcriptomics

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Gene expression profiling was performed by using Mouse Whole Genome Gene Expression BeadChip (Illumina). Samples included 7 high-LET radiation-induced HCC, 3 low-LET radiation-induced HCC, 4 spontaneous HCC, 10 non-HCC livers from high-LET–irradiated mice, 4 non-HCC livers from low-LET–irradiated mice and 6 normal livers from sham-irradiated mice. Each RNA sample, with 500 ng of total RNA, was amplified via the Ambion TotalPrep RNA amplification kit (ThermoFisher Scientific). This kit uses a T7 oligo(dT) primer to generate single-stranded cDNA, then a second strand synthesis to generate double-stranded cDNA, which is then column purified. We used in vitro transcription to synthesize biotin-labeled cRNA via T7 RNA polymerase. We then checked the column-purified cRNA for size and yield by using the Bio-Rad Experion system, and we hybridized 1.5 µg of cRNA to each array by using standard Illumina protocols with streptavidin-Cy3 (Amersham) for detection. Arrays were scanned on an Illumina Beadstation.

Ding L.H., Yu Y., Edmondson E.F., Weil M.M., Pop L.M., McCarthy M., Ullrich R.L, & Story M.D. (2021). Transcriptomic analysis links hepatocellular carcinoma (HCC) in HZE ion irradiated mice to a human HCC subtype with favorable outcomes. Scientific Reports, 11, 14052.

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Illumina Microarray Expression Analysis

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Fluorescence-labeled probes were prepared from an aliquot (250 ng) of total RNA from each time point following essential oil treatments. First strand cDNA was reverse transcribed from total RNA in T7-oligo-dT; and cRNA was synthesized by in vitro transcription from the T7 promoter and labeled with biotinylated UTP by the Illumina Total Prep RNA Amplification Kit (Ambion; Austin. TX, USA). The biotinylatedlabeled cRNA probes were hybridized overnight to Illumina human Ref-8 version 3 BeadChips containing probes representating a total of 24,526 transcripts (Illumina, San Diego, CA, USA). Following stringent washes, microarray chips were incubated with streptavidin-Cy3 (Amersham Biosciences; Piscataway, NJ, USA) and scanned on an Illumina BeadArray Reader (Illumina).

Dozmorov M.G., Yang Q., Wu W., Wren J., Suhail M.M., Woolley C.L., Young D.G., Fung K.M, & Lin H.K. (2014). Differential effects of selective frankincense (Ru Xiang) essential oil versus non-selective sandalwood (Tan Xiang) essential oil on cultured bladder cancer cells: a microarray and bioinformatics study. Chinese Medicine, 9, 18.

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RNA Integrity and Illumina Gene Expression Analysis

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The RNA quality was determined with the Agilent 2100 Bioanalyzer using RNA 6000 Nano Chips (Agilent Technologies). The RNA integrity numbers of the samples ranged from 9.1-10.
RNA labeling was performed on a Hamilton Star Roboter (Hamilton) using the Illumina Total Prep-96 RNA Amplification kit from Ambion (Applied Biosystems). Briefly, from 500 ng total RNA first and second strand cDNA was synthesized, and cleaned-up using Agencourt magnetic beads CleanKit (Beckman Coulter). After in vitro transcription, biotin-labeled cRNA was purified using Agencourt magnetic beads CleanKit. cRNA concentration was determined with Quant-iT RiboGreen (Invitrogen). cRNA (1.5 µg) was mixed and hybridized to Illumina Human HT-12 v3 BeadChips. All washing and staining steps were performed with a Little Dipper Processor for Illumina BeadChips from Scigene (Sunnyvale). Biotin was labeled with streptavidin-Cy3 (Amersham Biosciences).

Barucker C., Sommer A., Beckmann G., Eravci M., Harmeier A., Schipke C.G., Brockschnieder D., Dyrks T., Althoff V., Fraser P.E., Hazrati L.N., George-Hyslop P.S., Breitner J.C., Peters O, & Multhaup G. (2015). Alzheimer amyloid peptide aβ42 regulates gene expression of transcription and growth factors. Journal of Alzheimer's disease : JAD, 44(2).

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Cytogenetic Analysis of Chromosomes

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The C-banding method described in Badaeva et al. [1994] was used for analysis. Chromosomes were classified according to the nomenclature suggested in Friebe and Gill [1996] and Friebe et al. [1990] .
Fluorescence in situ Hybridization FISH was carried out according to a standard protocol [Badaeva et al., 1996b] with minor modifications [Zoshchuk et al., 2007] . The probes labelled with fluorescein were detected using anti-fluorescein/Oregon green, rabbit IgG fraction, Alexa Fluor 488 conjugate (Molecular Probes, USA). Biotin was detected with streptavidin-Cy3 (Amersham Pharmacia Biotech, USA). The slides were counter-stained with DAPI (4′,6-diamidino-2-phenylindole) in Vectashield mounting media (Vector laboratories, Peterborough, UK) and analyzed using a Zeiss Imager D-1 microscope. Selected metaphase cells were captured with an AxioCam HRm digital camera using software AxioVision, version 4.6. Images were processed in Adobe Photoshop, version CS5 (Adobe Systems, Edinburgh, UK). The FISH painted chromosomes were classified according to Komuro et al. [2013] and Megyeri et al. [2012] .

Badaeva E.D., Amosova A.V., Goncharov N.P., Macas J., Ruban A.S., Grechishnikova I.V., Zoshchuk S.A, & Houben A. (2015). A Set of Cytogenetic Markers Allows the Precise Identification of All A-Genome Chromosomes in Diploid and Polyploid Wheat. Cytogenetic and genome research, 146(1).

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