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Redivue α 32p dctp

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Redivue[α-32P] dCTP® is a radioactively labeled deoxycytidine triphosphate (dCTP) product. It is designed for use in various molecular biology applications that require radiolabeled nucleotides.

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Lab products found in correlation

Random prime labeling kit, by Roche (1 mentions) Biomax ms film, by Kodak (1 mentions) Quantity one quantitation software, by Bio-Rad (1 mentions)

3 protocols using redivue α 32p dctp

1

Cytokine Detection and Immune Response Analysis

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Example 20

Materials and Experimental Methods

Detection of Cytokines in Plasma.

Serum samples obtained from blood collected at different times during and after 7 daily lipofectin-complexed mRNA administrations are analyzed for mouse IFN-α, TNF-α, and IL-12 using ELISA kits.

Northern Blot Analysis.

Aliquots (2.0 μg) of RNA samples isolated from spleen are separated by denaturing 1.4% agarose gel electrophoresis, transferred to charged membranes (Schleicher and Schuell) and hybridized in MiracleHyb® (Stratagene). Membranes are probed for TNF-α, down-stream IFN signaling molecules (e.g. IRF7, IL-12 p35 and p40, and GAPDH) and other markers of immune activation. Specificity of all probes is confirmed by sequencing. To probe the membranes, 50 ng of DNA is labeled using Redivue[α-32P] dCTP® (Amersham) with a random prime labeling kit (Roche). Hybridized membranes are exposed to Kodak BioMax MS film using an MS intensifier screen at −70° C.

Histopathology.

Spleens from EPO-ψmRNA-treated and positive and negative control-treated mice are harvested, fixed, sectioned, stained with hematoxylin and eosin and examined by a veterinary pathologist for signs of immune activation.

US11801314B2. RNA containing modified nucleosides and methods of use thereof (2023-10-31). The Trustees of the University of Pennsylvania [US]. Inventors: Katalin Kariko [US], Drew Weissman [US].
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2

Quantitative RT-PCR Protocol for Plant Samples

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cDNA template preparation was performed as previously reported (Krogan et al., 2014 (link)). For
shoot samples, PCR reactions of cDNA template included 1.2μCi Redivue
[α-32P] dCTP (Amersham Biosciences,
Mississauga, ON, Canada) to facilitate product quantification. Low cycle numbers
(24 cycles) were used to prevent saturation of product amplification. Further,
at least two concentrations of template were amplified in parallel to ensure
that a doubling of the amount of starting template resulted in a proportional
doubling of the final PCR product. Electrophoresed RT-PCR products were scanned
by a Personal Molecular Imager FX Scanner and quantified by accompanying
Quantity One Quantitation Software (Bio-Rad). Root samples were analyzed
similarly, except that radioactive labeling was omitted and product intensity
was instead quantified by ImageJ software (National Institutes of Health, MD,
USA). Primer sequences are given in Supporting Information Table S1.
Krogan N.T., Marcos D., Weiner A.I, & Berleth T. (2016). The Auxin Response Factor MONOPTEROS controls meristem function and organogenesis in both the shoot and root through the direct regulation of PIN genes. The New phytologist, 212(1), 42-50.
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3

Purification and EMSA of His-MP(432) Protein

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The purification of His-MP(432) protein and EMSA experimental conditions
have been described (Krogan et
al., 2014). Labeled probes were created by PCR reactions
containing 20μCi of Redivue [α-32P]
dCTP (Amersham Biosciences). Primer sequences are provided in Table S2. The nonspecific
competitor used in EMSAs corresponded to −1870bp to −1729bp
(relative to translational start) of PIN3.
Krogan N.T., Marcos D., Weiner A.I, & Berleth T. (2016). The Auxin Response Factor MONOPTEROS controls meristem function and organogenesis in both the shoot and root through the direct regulation of PIN genes. The New phytologist, 212(1), 42-50.
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