Ultracruz autoradiography film

Manufactured by Santa Cruz Biotechnology

Sourced in United States

The UltraCruz Autoradiography Film is a photographic film used for the detection and visualization of radioactive signals in various biological and biochemical applications, such as autoradiography. The film is designed to capture and record the exposure of radioactive samples, allowing researchers to analyze and quantify the results of their experiments.

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12 protocols using ultracruz autoradiography film

Western Blot Protein Analysis Protocol

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Protein lysates (20-25μg) were denatured using 1X Laemmli Buffer and run on 7.5, 10, or 16% SDS PAGE gels at 100V for about 2 hours. Proteins were then transferred onto a PDVF membrane (BioRad or Thermo Fisher Scientific) at 100V for 1 hour. Ponceau-S staining was done to verify transfer. Non-specific binding was blocked by incubating the membrane for at least one hour at room temperature or overnight at 4°C in 5% milk in TBS for non-phosphorylated proteins, or 5% Bovine serum albumin (BSA Fraction V, Fisher Scientific) in TBS for phosphorylated proteins. The membrane was then blotted with primary antibody diluted in 5% BSA or Milk in TBS supplemented with 0.1% Tween 20 (TBST). After an overnight incubation at 4°C, the membrane was washed with TBST and incubated in the secondary antibody for at least 2 hours at room temperature in 5% BSA or Milk in TBST. After washing out the secondary antibody in TBST, the membrane was exposed to BioRad's Clarity Western ECL substrate according to manufacturer's instructions. Signal was detected using UltraCruz Autoradiography Film (Santa Cruz Biotech). Antibodies for GAPDH and p-IκBα were obtained from Cell Signaling (Catalog # 14C10 and 14D4, respectively). BCL2α, Pro-caspase 3, PARP-1, and cytochrome c antibodies were obtained from Santa Cruz Biotech Catalog # SC-7382, SC-7148, SC-8007, and SC-271627, respectively.

Alexander-Savino C.V., Hayden M.S., Richardson C., Zhao J, & Poligone B. (2016). Doxycycline is an NF-κB inhibitor that induces apoptotic cell death in malignant T-cells. Oncotarget, 7(46), 75954-75967.

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Western Blot Quantification Protocol

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Tissues from organs were homogenized using a glass Dounce homogenizer and lysed in RIPA buffer containing protease and phosphatase inhibitors. Total protein concentrations of lysed samples were determined by using Pierce^TM Coomassive (Bradford) Protein Assay Kit (Thermo Fisher Scientific Inc., MA, USA). 20 μg of the protein lysates was electrophoresed on SDS-PAGE gel and transferred onto nitrocellulose membranes. After blocking with 5% non-fat dry milk dissolved in TBST for 1 hour, the membranes were probed with a given primary antibody overnight at 4°C, reacted with HRP-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody and were detected by using the Miracle Star^TM Femto Western Blot Detection System (Intron Biotechnology Co., Ltd., Gyeonggi-do, Korea) and X-ray film (UltraCruz® Autoradiography Film, Santa Cruz Biotechnology Inc., TX, USA) or iBright CL1000 Imaging System (Thermo Fisher Scientific Inc., MA, USA). Quantification of blots (densitometry) was performed using NIH ImageJ software.

Nguyen L.V., Ta Q.V., Dang T.B., Nguyen P.H., Nguyen T., Pham T.V., Nguyen T.H., Baker S., Le Tran T., Yang D.J., Kim K.W, & Doan K.V. (2019). Carvedilol improves glucose tolerance and insulin sensitivity in treatment of adrenergic overdrive in high fat diet-induced obesity in mice. PLoS ONE, 14(11), e0224674.

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Quantifying Extracellular Vesicles in Urine

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Western blotting was performed as described 27 (link). For detection, x-ray film (Ultra Cruz™ Autoradiography Film, Santa Cruz Biotechnology (SC), Dallas, TX, USA) or, alternatively, Odyssey infrared scanner (LI-COR® Biosciences Ltd., Cambridge, UK) were used. To quantify the EVs for the metabolomics study (Fig. 2A), EVs from equal volumes of urine were loaded to gels. The CD9 bands were quantified by Image J 28 (link). Proteins from samples depicting the purification process (Fig. 1G, 1800 g and 8000 g urine supernatant and filtrate samples), were extracted with ProteoSpin™ Urine Protein Concentration Micro Kit (Norgen Biotek Corp., Ontario, Canada). Protein concentrations were measured with BradfordUltra reagent (Expedeon) and 2 µg of protein loaded to gels from all purification process samples (Fig. 1G) and 20 µg of LNCaP cell lysate control. Platelet EVs and platelets from equal volumes of metabolomics samples were loaded to gels (Fig. S1).

Puhka M., Takatalo M., Nordberg M.E., Valkonen S., Nandania J., Aatonen M., Yliperttula M., Laitinen S., Velagapudi V., Mirtti T., Kallioniemi O., Rannikko A., Siljander P.R, & af Hällström T.M. (2017). Metabolomic Profiling of Extracellular Vesicles and Alternative Normalization Methods Reveal Enriched Metabolites and Strategies to Study Prostate Cancer-Related Changes. Theranostics, 7(16), 3824-3841.

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Western Blot Protein Expression Analysis

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For detecting the protein expression, RIPA lysis buffer (Beyotime, China) was first used to isolate the total proteins from cells. Then, the isolated proteins were separated by 10% SDS‐PAGE followed by transferring onto PVDF membranes (Beyotime, China). Next, the membranes after blocking with fat‐free milk were incubated with anti‐FNDC3B (Cat#: sc‐393997, Santa Cruz Biotechnology) or anti‐GAPDH (Cat#: sc‐365062, Santa Cruz Biotechnology) followed by incubating with anti‐mouse IgG‐HRP (Cat#: sc‐2005, Santa Cruz Biotechnology). Finally, the protein was visualized using Western Blotting Luminol Reagent (Cat#: sc‐2048, Santa Cruz Biotechnology) and UltraCruz Autoradiography Film (Cat#: sc‐201696, Santa Cruz Biotechnology).

Zhang J., Peng Y., Jiang S, & Li J. (2022). Hsa_circRNA_0001971 contributes to oral squamous cell carcinoma progression via miR‐186‐5p/Fibronectin type III domain containing 3B axis. Journal of Clinical Laboratory Analysis, 36(3), e24245.

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Western Blot Analysis of Proteins

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Previously described techniques were used (Aravamudan et al., 2017 (link)). Briefly, cells were washed, sonicated in lysis buffer (Cell Signaling Technologies, Beverly, MA, USA) containing protease and phosphatase inhibitors, and resultant supernatants were assayed for total protein content using the DC Protein Assay kit (BioRad, Hercules, CA, USA). 30 μg equivalent protein from each lysate were loaded on 10% SDS-page and transferred onto 0.22 μm PVDF membranes. Non-specific binding was blocked using 5.0% bovine serum albumin (BSA) and the membranes were probed overnight at 4°C with specific antibodies of interest. Blots were then incubated with HRP-conjugated secondary antibodies. β-actin was used as loading control. Protein expression was detected using Luminol Reagent (Santa Cruz, then exposed to UltraCruz Autoradiography Film (Santa Cruz), and scanned for densitometry analysis using ImageJ 1.50i software with integrated density values normalized for loading control and phosphorylated proteins. The ratios obtained for western blot analysis were first normalized by dividing the raw values of proteins of interest with the raw values of either β-Actin or the total protein. The obtained values were then normalized to vehicle.

Ambhore N.S., Katragadda R., Kalidhindi R.S., Thompson M.A., Pabelick C.M., Prakash Y, & Venkatachalem S. (2018). Estrogen Receptor Beta Signaling Inhibits PDGF Induced Human Airway Smooth Muscle Proliferation. Molecular and cellular endocrinology, 476, 37-47.

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Western Blot Protein Quantification

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Total cell proteins and the cytoplasmic and nuclear fractions were obtained from 70% confluent cell cultures. Western blotting (WB) was performed as previously described [95 (link)]. Blots were incubated with primary antibodies (overnight, 4 °C) and then with appropriate horseradish-peroxidase-conjugated secondary antibodies (1 h, room temperature). Immunoreactive bands were detected using the ECL Western blotting detection system (Santa Cruz Biotechnology (Dallas, TX, USA), sc-2048). Images were captured using UltraCruz Autoradiography Film (Santa Cruz Biotechnology) or iBright Imaging System (Thermo-Fisher (Waltham, MA, USA). The images from films were acquired using an Epson Perfection scanner (Epson, Japan) using Photoshop software (Adobe) (https://www.adobe.com/au/products/photoshop.html, accessed on 20 July 2023). The optical densities of the spots were analyzed by using ImageJ software (NIH) (https://imagej.en.softonic.com/download, accessed on 20 July 2023).

Morelli C., Chiodo C., Nocito M.C., Cormace A., Catalano S., Sisci D., Sirianni R., Casaburi I., Andò S, & Lanzino M. (2023). Androgens Modulate Bcl-2 Agonist of Cell Death (BAD) Expression and Function in Breast Cancer Cells. International Journal of Molecular Sciences, 24(17), 13464.

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Urinary Extracellular Vesicle Characterization

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Urinary EV samples were analyzed as explained previously.²² (link)^,⁶⁶ (link) Briefly, Western blotting of uEV marker proteins was performed using antibodies against CD9 (SC-13118, Santa Cruz) and podocalyxin (clone 3D3, Novus Biologicals). EV from equal volumes of urine (1.7 ml) and 20 μg of protein from LNCaP cell lysate control were loaded to gels. For detection, x-ray film (Ultra Cruz™ Autoradiography Film, Santa Cruz Biotechnology (SC), Dallas, TX, USA) was used.
Electron microscopy (EM) samples were prepared with a negative staining protocol. The samples were viewed with Jeol JEM-1400 (Jeol Ltd., Tokyo, Japan) at 80 kV and imaged with Gatan Orius SC 1000B CCD-camera (Gatan Inc., USA) with 4008 × 2672 px image size and no binning.
NTA was done with Nanosight LM14 (Malvern Instruments Ltd, Malvern, UK) with blue (404 nm, 70 mW) laser and SCMOS camera (Hamamatsu photonics K.K., Hamamatsu, Japan). Samples were diluted in filtered (0.1μm, Millex VV, Millipore) DPBS to obtain 40-100 particles per view. Five 30 s videos were recorded with camera level 13. The videos were analyzed using software version 3.0 (Malvern Instruments Ltd), detection threshold 5 and screen gain 10.

Dwivedi O.P., Barreiro K., Käräjämäki A., Valo E., Giri A.K., Prasad R.B., Roy R.D., Thorn L.M., Rannikko A., Holthöfer H., Gooding K.M., Sourbron S., Delic D., Gomez M.F., Groop P.H., Tuomi T., Forsblom C., Groop L, & Puhka M. (2023). Genome-wide mRNA profiling in urinary extracellular vesicles reveals stress gene signature for diabetic kidney disease. iScience, 26(5), 106686.

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Western Blot Protein Analysis Protocol

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Protein lysates were generated using radioimmunoprecipitation buffer supplemented with protease inhibitors cocktail (Complete, Roche), total protein content was determined using BCA Protein Assay Kit (Pierce), and 5–20 μg of total protein was resolved using Any-kD Mini-PROTEAN TGX Precast SDS page gels (Biorad). Gels were blotted onto nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Biorad) and blocked with 5% non-fat milk powder in PBS with 0.01% of Tween (PBST). Membranes were incubated with primary antibodies overnight at 4 °C, thereafter appropriate HRP-labeled secondary antibodies were incubated for 1 hour at room temperature and thoroughly washed with PBST. Next, membranes were incubated with SuperSignal West Dura Chemiluminescent Substrate (Pierce) and exposed on BioMax XAR Film (Kodak) or Ultracruz autoradiography film (Santa Cruz).

de Bruin R.G., van der Veer E.P., Prins J., Lee D.H., Dane M.J., Zhang H., Roeten M.K., Bijkerk R., de Boer H.C., Rabelink T.J., van Zonneveld A.J, & van Gils J.M. (2016). The RNA-binding protein quaking maintains endothelial barrier function and affects VE-cadherin and β-catenin protein expression. Scientific Reports, 6, 21643.

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Immunoprecipitation and Immunoblotting of N-WASp

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Cell lysates were immunoprecipitated with the mouse polyclonal anti-SNX9 antibody (Santa Cruz) and SureBeads Protein G Magnetic Beads (Bio-Rad Laboratories, Hercules, CA, USA) for 1 h at room temperature. Immunoprecipitation using SureBeads was performed in accordance with the manufacturer’s instructions. The samples were electrophoresed through a reducing SDS polyacrylamide gel and electroblotted onto a nitrocellulose membrane. The membranes were blocked and incubated with the antibody to detect N-WASp (MilliporeSigma, Burlington, MA, USA) using horseradish peroxidase-linked secondary antibodies and an Enhanced Chemiluminescence System (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Autoradiography was performed using UltraCruz Autoradiography Films (Santa Cruz Biotechnology). The developed films were scanned and the optical densities of the protein bands were quantified using NIH ImageJ.

Gychka S.G., Shults N.V., Nikolaienko S.I., Marcocci L., Sariipek N.E., Rybka V., Malysheva T.A., Dibrova V.A., Suzuki Y.J, & Gavrish A.S. (2020). Vasa Vasorum Lumen Narrowing in Brain Vascular Hyalinosis in Systemic Hypertension Patients Who Died of Ischemic Stroke. International Journal of Molecular Sciences, 21(24), 9611.

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Western Blot Analysis of Signaling Proteins

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Cell lysates and protein gel electrophoresis samples were prepared as previously described [11 (link)]. Equal protein amounts of samples were electrophoresed through a reducing SDS polyacrylamide gel and electroblotted onto a nitrocellulose membrane. Membranes were blocked and incubated with antibodies for pRaf-1 (S338; Catalog # 9427), pERK1/2 (T202/Y204; Catalog # 4370), pMEK1/2 (S217/221; Catalog # 9154), pStat3 (Y705; Catalog # 9145), phospho-threonine (Catalog # 9381), ERK1/2, MEK1/2 (Cell Signaling Technology, Danvers, MA, USA), neurotensin receptor (NTR)-1, NTR2, NTR3 and glutaraldehyde-3-phosphate-dehydrogenase (G3PDH) (Santa Cruz Biotechnology). Levels of proteins were detected by using horseradish peroxidase-linked secondary antibodies and an Enhanced Chemiluminescence System (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Autoradiography was performed using UltraCruz Autoradiography Films (Santa Cruz Biotechnology). The developed films were scanned and optical densities of protein bands were quantified using NIH ImageJ.

Shults N.V., Almansour F.S., Rybka V., Suzuki D.I, & Suzuki Y.J. (2018). Ligand-mediated dephosphorylation signaling for MAP kinase. Cellular signalling, 52, 147-154.

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