Autoradiography film

Manufactured by Thermo Fisher Scientific

Sourced in United States

Autoradiography film is a type of photographic film used to visualize and detect radioactive signals in biological samples. It is a sensitive medium that captures the radiation emitted from radioactive labels, such as isotopes, allowing researchers to analyze the distribution and intensity of these signals within their experiments.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Pvdf membrane, by Bio-Rad (3 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Supersignal west pico chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions) Un scan it gel version 5, by Silk Scientific (1 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Complete mini protease inhibitor cocktail tablet, by Roche (1 mentions) Supersignal west pico, by Thermo Fisher Scientific (1 mentions) Tubulin, by Cell Signaling Technology (1 mentions) Cebpd, by Santa Cruz Biotechnology (1 mentions) C myc, by Cell Signaling Technology (1 mentions) Bca assay, by Bio-Rad (1 mentions) Ripa buffer, by Cell Signaling Technology (1 mentions) Protease inhibitor cocktail tablet, by Roche (1 mentions) Rabbit anti histone h3, by Cell Signaling Technology (1 mentions) Histone extraction kit, by Abcam (1 mentions) Rabbit anti h3k27ac, by Abcam (1 mentions) Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions) Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions) Anti mouse antibody, by GE Healthcare (1 mentions)

7 protocols using autoradiography film

Western Blot Protein Detection

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Cells were lysed in Pierce IP Lysis Buffer (Pierce) supplemented with Halt Protease Inhibitor Cocktail (Thermo Scientific) or Complete mini protease inhibitor cocktail tablets (Roche), and the resulting lysates were separated by 10% SDS-PAGE and then electroblotted onto Invitrolon PVDF membranes (Novex, Life Technologies). Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline-Tween 20 (0.1%) buffer and then probed with primary antibody in 5% BSA-Tris-buffered saline-Tween 20 (0.1%) buffer. The antigen-antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibody to rabbit or mouse IgG (Cell Signaling Technology) with SuperSignal West Pico Chemiluminescent Substrate Kit (Thermo Scientific) and the images were captured by exposing membranes to autoradiography films (GeneMate). The films were scanned and specific signals were quantified by UN-SCAN-IT gel Version 5.3 (Silk Scientific).

Tam S.Y., Lilla J.N., Chen C.C., Kalesnikoff J, & Tsai M. (2015). RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells. PLoS ONE, 10(11), e0142935.

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Quantitative Immunoblotting Analysis of Glutamate Transporters

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Immunoblotting procedures were done as previously described (Sari et al., 2010 ; Sari et al., 2009 (link)). In brief, striatum and hippocampus samples were lysed in lysis buffer. Quantified proteins were separated through 10% polyacrylamide gels in 1× laemmli buffer. Proteins were then transferred onto PVDF membrane (Bio-Rad Laboratories). Membranes were further blocked with 3 % blocking buffer (fat free milk, LabScientific) in 1× TBST. The membranes were then incubated with primary antibodies (GLT-1, xCT or GLAST) overnight at 4°C. β-tubulin was used as a loading control. Membranes were incubated with chemiluminescent substrate (Super Signal West Pico, Thermo Scientific) and exposed to autoradiography films (Denvilla Scientific Inc.). The membranes were then developed using X-Ray film processor (Konica SRX101A – Tabletop). Images of immunoblots were quantified using MCID Digital Imaging Software.

Alshehri F.S., Althobaiti Y.S, & Sari Y. (2016). Effects of Administered Ethanol and Methamphetamine on Glial Glutamate Transporters in Rat Striatum and Hippocampus. Journal of molecular neuroscience : MN, 61(3), 343-350.

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Protein Expression Profiling of NF-kB Pathway

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Cells were lysed in RIPA buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail tablets (Roche Diagnostics). Total protein concentration of the lysates was measured by BCA assay (Bio-Rad), and equal amounts of protein were loaded on 4% to 12% PAGE gels (Life Technologies). PVDF membranes were blocked with 5% nonfat dried milk in TBS buffer and probed with antibody for p65, p-p65, p50, IkBα, c-Myc, PARP, REL, RELB, tubulin (Cell Signaling Technology), CEBPD, and SOD2 (Santa Cruz Biotechnology). Signals from secondary antibodies were detected using ECL (Pierce) and auto-radiography films (Thermo Fisher Scientific). Antibodies for IHC (p-p65 and CEBPD) were from Abcam.

Ambrosini G., Do C., Tycko B., Realubit R.B., Karan C., Musi E., Carvajal R.D., Chua V., Aplin A.E, & Schwartz G.K. (2019). Inhibition of NF-κB–Dependent Signaling Enhances Sensitivity and Overcomes Resistance to BET Inhibition in Uveal Melanoma. Cancer research, 79(9), 2415-2425.

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Histone Extraction and Western Blot

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Whole retina was dissected and homogenized in a dounce homogenizer and histones isolated with the Histone Extraction Kit (ab113476, Abcam). Extracts were run on a 4–20% gel (Bio-Rad) before blotting. PVDF membranes (Bio-Rad) were then blocked overnight at 4 °C in a milk blocking solution (5% powdered milk, 0.5% Triton X-100, 0.1% NaN₃ in PBS). Following three 30-min washes in a wash buffer (0.5% Tween in PBS), membranes were incubated with primary antibodies in milk blocking solution overnight at 4 °C. Membranes were then washed three times with wash buffer and incubated with at room temperature with 1:10,000 rabbit anti-HRP in TBS-T for 2 h. Following a final wash step, membranes were developed with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and exposed on Autoradiography Film (Blue Devil West). All blots were scanned at 600 DPI and quantified in ImageJ (NIH, Bethesda, MD) by transforming the image to 8-bit format and quantifying the grey value via Uncalibrated OD in a region of interest drawn around histone bands.
Primary antibodies: rabbit anti-Histone H3 (Cell Signaling, 1:10,000), rabbit anti-H3K27ac (Abcam, 1:10,000).

Jorstad N.L., Wilken M.S., Grimes W.N., Wohl S.G., VandenBosch L.S., Yoshimatsu T., Wong R.O., Rieke F, & Reh T.A. (2017). Stimulation of functional neuronal regeneration from Müller glia in adult mice. Nature, 548(7665), 103-107.

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Western Blot Analysis of Hypoxia Signaling

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BMDMs were washed on ice with ice cold PBS, then lysed over 30 minutes with ice cold RIPA Lysis Buffer containing a cocktail of protease inhibitors (Roche) and phosphatase inhibitors (NEB) with gentle agitation every 10 minutes. Cellular debris was clarified (13,000g, 10 minutes, 4°C). Reducing agent (NuPAGE Thermo) and Sample Buffer was added to supernatant, and boiled at 95°C for 10 minutes. Protein lysates were resolved on NuPAGE™ 4–12% Bis-Tris Protein Gels using MOPS running buffer (Invitrogen), transferred to nitrocellulose membrane, and blocked with 5% OmniBlok™ Dry Milk in PBS. Detection of HIF1α (D2U3T), Hif2α (D9E3), phospho-c-JUN (54B3 and 9164), phospho-JNK (81E11), total c-JUN (60A8) and total JNK (9252) was conducted using monoclonal antibodies (Cell Signaling). β-tubulin (E7) was detected using a monoclonal antibody (DSHB). HRP-conjugated anti-rabbit (Cell Signaling, 7074) or anti-mouse antibody (GE Healthcare, NA931V) was used as secondary antibodies and detected using SuperSignal West Pico PLUS Chemiluminiscent Substrate (Thermo) and autoradiography film (Denville).

Solis A.G., Bielecki P., Steach H.R., Sharma L., Harman C.C., Yun S., Palm N.W., de Zoete M.R., Warnock J.N., To S.D., York A.G., Mack M., Schwartz M.A., Cruz C.S., Jackson R, & Flavell R.A. (2019). Mechanosensation of Cyclical Force by PIEZO1 is Essential for Innate Immunity. Nature, 573(7772), 69-74.

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Whole Tissue/Cell Extract Preparation and Western Blot Analysis

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For whole tissue/cell extract preparation, tissues and cells were lysed in Pierce™ RIPA buffer (Thermo Fisher Scientific, Waltham, MA, U.S.A.) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Homogenates were centrifuged, and supernatants were used for protein measurement via Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Western blot samples were prepared in Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA, U.S.A.). They were loaded onto a 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gel and transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, U.S.A.). The membrane was blocked, and antibodies diluted in 5% nonfat dry milk (Bio-Rad Laboratories, Hercules, CA, U.S.A.) or in 5% bovine serum albumin (Irvine Scientific, Santa Ana, CA, U.S.A.) were used. Protein bands were visualized using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, U.S.A.) and developed on the autoradiography film (Santa Cruz, Dallas, TX, U.S.A.).

Ristic B., Sivaprakasam S., Narayanan M, & Ganapathy V. (2020). Hereditary hemochromatosis disrupts uric acid homeostasis and causes hyperuricemia via altered expression/activity of xanthine oxidase and ABCG2. Biochemical Journal, 477(8), 1499-1513.

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Expression of HA-tagged RGS2 in HEMs

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Expression of HA-tagged RGS2 in HEMs was confirmed via Western blotting. Cells were homogenized ≥10 d after infection in ice-cold RIPA buffer (Thermo Fisher Scientific) containing protease inhibitor cocktail (Roche). Samples were agitated at 4°C for 30 min and then centrifuged at 16,000 g for 30 min at 4°C. Protein content was determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Equal amounts of protein were loaded onto each lane, separated by electrophoresis on NuPAGE Bis-Tris gels (Invitrogen), and transferred to PVDF membranes (Roche). Membranes were blocked at room temperature for 1 h and incubated overnight at 4°C with rat monoclonal anti-HA antibody clone 3F10 (1:500; Roche), followed by 1 h at room temperature with HRP-conjugated goat anti–rat IgG affinity-purified antibody (1:5,000; EMD Millipore). Antibodies were detected using the SuperSignal West Femto enhanced chemiluminescence system (Thermo Fisher Scientific) and imaged using autoradiography film (Thermo Fisher Scientific).

Bellono N.W., Najera J.A, & Oancea E. (2014). UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes. The Journal of General Physiology, 143(2), 203-214.

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