Autoradiography film

Manufactured by Merck Group

Sourced in United Kingdom, United States

Autoradiography film is a type of photographic film used to detect and visualize radioactive signals in biological samples. It is a sensitive imaging medium that captures the radiation emitted from radioactive isotopes, allowing researchers to analyze the distribution and abundance of specific molecules or proteins within a sample.

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Lab products found in correlation

Bca kit, by Thermo Fisher Scientific (3 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Anti actin, by Merck Group (1 mentions) Immobilon ecl western blotting detection kit reagent, by Merck Group (1 mentions) Anti cyclin e he12, by Santa Cruz Biotechnology (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Rabbit anti p p38, by Merck Group (1 mentions) Mouse anti gapdh, by Merck Group (1 mentions) Enhanced chemi luminescence (ecl), by Merck Group (1 mentions) Luminata forte western hrp substrate, by Merck Group (1 mentions) Pvdf membrane, by Merck Group (1 mentions)

4 protocols using autoradiography film

Western Blot Analysis of Cyclin E

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Protein extracts were prepared using RIPA buffer supplemented with 1% protease inhibitor cocktail (Thermo Fisher Scientific Inc.), 1% phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc.) and quantified by BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membrane (Bio-Rad Laboratories). After, membranes were blocked for 1h with 5% skim milk in 0.1% Tween PBS and incubated with the specific primary antibodies overnight at 4°C. Then, membranes were treated with the appropriate secondary antibody for 1 h at room temperature. All blots were treated with Immobilon ECL Western Blotting Detection Kit Reagent (EMD Millipore) and developed after exposure to an autoradiography film (VWR International). The following antibodies were used: anti-cyclin E (HE12, Santa Cruz) and anti-actin (691001, Millipore).

Benito A., Polat I.H., Noé V., Ciudad C.J., Marin S, & Cascante M. (2017). Glucose-6-phosphate dehydrogenase and transketolase modulate breast cancer cell metabolic reprogramming and correlate with poor patient outcome. Oncotarget, 8(63), 106693-106706.

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Western Blot Analysis of p-p38 and GAPDH

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To identify the expression of p-p38 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), rats were sacrificed by decapitation and the lumbosacral spinal cord was promptly removed, immediately frozen in liquid nitrogen, stored at -80° C prior to processing, and homogenized in lysis buffer. Forty-five μg of protein was separated on a 10% SDS-PAGE gel and blotted onto a nitrocellulose membrane. The membranes were blocked with 10% nonfat dry milk and then incubated overnight at 4°C with rabbit anti-p-p38 (1:100; Merck Millipore, USA) and mouse anti-GAPDH (1:2000; Merck Millipore). Immunoreactive bands were detected using secondary antibodies and ECL (Merck Millipore), followed by exposing the membrane to autoradiography film (GE Healthcare Limited, UK).

Horst A., de Souza J.A., Santos M.C., Riffel A.P., Kolberg C., Ribeiro M.F., de Fraga L.S, & Partata W.A. (2017). N-acetylcysteine downregulates phosphorylated p-38 expression but does not reverse the increased superoxide anion levels in the spinal cord of rats with neuropathic pain. Brazilian Journal of Medical and Biological Research, 50(2), e5801.

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Detection of PrPres Accumulation

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PrP^res accumulation was also evaluated by immunoblot, to confirm the molecular weight and glycoform pattern. At P-6, cells were collected by lifting and centrifugation at 1,000 × g (JA50.5 rotor) for 7 minutes at room temperature (RT). Cells were washed with 1× Dulbecco’s-PBS (D-PBS) and pellets were frozen at −80 °C until testing. Cell pellets were lysed and lysates were electrophoresed, electroblotted, and immunoblotted as previously described (Stanton et al., 2008 (link)). Briefly, lysates were measured for total protein by the bicinchoninic acid protein assay (BCA kit; Thermo Scientific) and diluted to equivalent protein concentrations prior to proteinase K (50 μg μl⁻¹, 2.5 units mg⁻¹, Roche Applied Science) digestion and phosphotungstic acid (PTA) precipitation; both steps included a 1-hour incubation at 37 °C. PrP^res bands were labeled using 3.5 μg ml⁻¹ of the mAb F99/97.6.1 (O’Rourke et al., 2000 (link)) and a goat anti-mouse IgG₁ antibody conjugated to HRP (1:5000; Southern Biotechnology Associates). Membranes were incubated with chemiluminescent substrate (Luminata^™ Forte Western HRP Substrate; Millipore) for 3 minutes and signals were detected by capture onto autoradiography film (GeneMate, Kaysville, UT, USA).

Dinkel K.D., Schneider D.A., Muñoz-Gutiérrez J.F., McElliott V.R, & Stanton J.B. (2017). Correlation of Cellular Factors and Differential Scrapie Prion Permissiveness in Ovine Microglia. Virus research, 240, 69-80.

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Quantitative HMT Assay for PRC2 Activity

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Standard HMT assays were performed in a total volume of 15 µl containing HMT buffer (50 mM Tris-HCl, pH 8.5, 5 mM MgCl₂, and 4 mM DTT) with 500 nM of ³H-labeled S-Adenosylmethionine (SAM, Perkin Elmer), 300 nM of nucleosomes, and recombinant human PRC2 complexes under the following conditions. The reaction mixture was incubated for 60 min at 30 °C and stopped by adding 4 µl of STOP buffer (0.2 M Tris-HCl, pH 6.8, 20 % glycerol, 10% SDS, 10 mM β-mercaptoethanol, and 0.05 % Bromophenol blue). A titration of PRC2 (from 5 to 60 nM) was performed under these conditions in order to optimize the HMT reaction within a linear range, and the yield of each HMT reaction was measured using the following procedures. After HMT reactions, samples were incubated for 5 min at 95 °C and separ ated on SDS-PAGE gels. The gels were then subjected to Coomassie blue staining for protein visualization or wet transfer of proteins to 0.45 µm PVDF membranes (Millipore) and exposed to autoradiography film (Denville scientific).

Lee C.H., Holder M., Grau D., Saldaña-Meyer R., Yu J.R., Ganai R.A., Zhang J., Wang M., LeRoy G., Dobenecker M.W., Reinberg D, & Armache K.J. (2018). Distinct stimulatory mechanisms regulate the catalytic activity of Polycomb Repressive Complex 2 (PRC2). Molecular cell, 70(3), 435-448.e5.

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