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X ray film

Manufactured by AGFA HealthCare
Sourced in Belgium, United Kingdom, United States, Germany

X-ray film is a type of photographic film used in medical imaging to capture and record radiographic images. It is designed to be sensitive to X-ray radiation, allowing the creation of detailed images of the body's internal structures.

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94 protocols using x ray film

1

Western Blot Analysis of Proteins

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Cells were lysed in IP buffer (50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1% NP40, 2 mM Sodium orthovanadate, 10 mM NaF, 10 mM β-glycerophosphate, 1 mM PMSF) [15 (link)]. Proteins in lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to PVDF membrane (Millipore). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG, sheep anti-mouse IgG, and donkey anti-goat IgG were used as secondary antibodies. Chemiluminescence detection was performed using WEST-ZOL plus (iNtRON) and West Femto Substrate (Thermofisher). X-ray films (Agfa) and LAS (Image Quant; LAS4000) were used to visualize the chemiluminescence signal. 0.1% TBS-T was used for washing and blocking solution preparation, and 0.1% PBS-T was used for LC3B blotting [51 (link), 52 (link)].
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2

EPEC Protein Detection by Western Blot

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Sodium Dodecyl Sulfate–Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were performed as reported previously [58 (link)]. Preparation of EPEC protein extracts are described in the S1 Text. For detection of EPEC proteins by Western blotting, membranes were incubated with primary rabbit antibodies anti-EspB (1:2000), anti-EscC (1:1000), anti-EscJ (1:5000), anti-EscD (1:1000) and anti-Intimin280 (1:5000). Use of polyclonal rabbit sera against EPEC Intimin-280, EscC and EscD were described previously [57 (link), 59 (link)]. Rabbit polyclonal serum against EscJ and EspB was a kind gift of Dr. Bertha González-Pedrajo (UNAM, Mexico). Bound rabbit antibodies were detected with secondary Protein A-peroxidase (POD) conjugate (Life Technologies, 1:5000). GroEL was detected with mAb anti-GroEL-POD conjugate (1:5000; Sigma). Membranes were developed by chemiluminiscence using the Clarity Western ECL Substrate kit (Bio-Rad). The membranes were then developed by exposure to X-ray films (Agfa) or with a Fuji LAS 3000 image when the signal was quantified.
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3

Immunoblotting Analysis of Akt and Foxo

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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and penicillin/streptomycin were purchased from GIBCO (Thermo Fisher Scientific, Waltham, MA, USA). Anti-phospho-Akt, anti-total Akt, anti-phospho-Foxo and anti-Foxo antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-mouse HRP and anti-rabbit HRP were purchased from Thermo Scientific. PVDF membranes and SuperSignal® West Pico and West Femto Chemiluminescent Substrate for western blotting analysis were purchased from Thermo Scientific, and X-ray films from AGFA (Mortsel, Belgium). SYBR Green Master Mix for the real-time PCR analysis was purchased from Applied Biosystems (Foster City, CA, USA).
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4

Protein Extraction and Western Blot Analysis

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The cell lysates were prepared as described previously [20 (link)]. Briefly, the Tris-based buffer containing phosphatase inhibitors and protease inhibitors (Roche, Basel, Switzerland) was applied and cleared with centrifugation. The protein in soluble fractions was quantified using a BCA kit (Thermo) and standard western blotting was performed using PVDF membrane (Merk Millipore, Boston, MA, USA). The antibodies used were as follows: α-tubulin (sc-23948) and PARP1/2 (sc-7150) were purchased from Santa Cruz (Santa Cruz, CA, USA). SCD1 (ab19862) was purchased from Abcam (Cambridge, UK). Cleaved active caspase-3 (#9661) was purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies conjugated with horseradish peroxidase were purchased from Cell Signaling Technology. The signals were visualized using SuperSignal® Femto (Thermo) and X-ray films (Agfa, Mortsel, Belgium).
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5

Detecting Actin Proteins by Western Blot

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The proteins were separated depending on their molecular weight in SDS-PAGE using 12.5% acrylamide gels. The proteins were transferred on a nitrocellulose membrane using semi-dry blot chamber. Subsequent to Ponceau S (AppliChem GmbH, Darmstadt, Germany) staining, unspecific binding was blocked by incubation in 5% skim milk powder diluted in PBS-T (137 mM NaCl, 2.7 mM KCl 8 mM Na2HPO4, 1.8 mM KH2PO4, 0.1% Tween®20, pH 7.4) for 1 h at RT. The membrane was quickly washed with PBS-T and incubated with actin monoclonal antibody (1:10,000, ACTN05(C4); Thermo Fisher Scientific, Waltham, MA, USA) or streptavidin-peroxidase conjugate (1:5000; Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS-T for 1 h at RT. Afterwards, unbound proteins were removed by washing three times with PBS-T for 5 min at RT on an orbital shaker. For the detection of actin, the membrane was further incubated with the secondary goat anti-mouse antibody-HRP (1:2500; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at RT and washed three times with PBS-T. The peroxidase marked proteins were detected using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and X-ray films (AGFA Health Care, Mortsel, Belgium).
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6

Western Blot Analysis of Granulosa Cells

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Granulosa cells were lysed with RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-X 100) containing 1X protease inhibitor cocktail (Roche, Basel, Switzerland) and resolved on 10% polyacrylamide-SDS gel. Following electrophoresis, proteins were transferred to PVDF membrane (Thermo Scientific, Rockford, IL, USA) and blocked with TBST (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20) containing 5% non-fat milk. The membrane was incubated overnight at 4 °C with anti-Ahr (SA-210), anti-p-CREB and anti-actin antibodies in TBST milk. ReBlot Plus Strong Antibody Stripping Solution (Merck Serono GmbH, Darmstadt, Germany) was used for stripping (anti-p-CREB) and re-blotting (anti-actin). Goat anti-rabbit secondary IgG-HRP and Immobilon Western chemiluminescent HRP substrate (Millipore, Billerica, MA, USA) were used to detect signals on X-ray films (Agfa, Mortsel, Belgium). The protein bands were quantified using ImageJ software.
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7

Rapamycin-Induced Transcriptional Changes

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Northern blot analysis was performed as described previously [5 (link)]. In short, yeast cultures were grown overnight on YPD. Cultures were then diluted and allowed to grow till an OD600nm of 1.5. Then control samples were taken (-30 and -15 min). Next, rapamycin was added to a final concentration of 200 nM and samples were taken after 15, 30, 60, and 120 min. RNA extraction and Northern blotting were performed as described previously [43 (link)]. The filters were hybridized with 32P-dCTP-labelled probes, generated with the High Prime kit (Roche, Merck, Hoeilaart, Belgium). Primers used for generation of the probes are listed in S3 Table. After washing, the filters were exposed to X-ray films (AGFA, Mortsel, Belgium).
For RT-PCR for PHO5 expression analysis, 300 ng of the total RNA was retro-transcribed using the first-strand cDNA Synthesis kit (Nzytech, Lissabon, Portugal). NZYSpeedy qPCR Green Master Mix SYBR green Master Mix (Nzytech, Lisssabon, Portugal) was used to perform quantitative PCR in an Applied Biosystems 7500 fast qPCR system (Merck Life Sciences, Algés, Portugal). Data were analyzed with the Δ2CT method and normalized to the expression of ACT1, PDA1 and TDH2 genes in the same sample. The primer pairs used are listed in S3 Table.
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8

Quantitative Western Blot Analysis

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For protein quantification, the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, USA) was used. After addition of 4x sample buffer (50 mM Tris, pH 6.8, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 0.0125% bromophenol blue, and 1% glycine), 50 μg of total cell lysate per lane was subjected to 7.5% SDS-PAGE and transferred onto a nitrocellulose membrane (0.2 μm pore size; Schleicher & Schuell Microscience, Dassel, Germany). The efficiency of protein transfer and equal loading was confirmed by staining the membrane with Ponceau S (Sigma Aldrich, Munich, Germany). Membranes were blocked overnight at 4°C with 5% (w/v) nonfat milk in TBS-T (10 mM Tris, pH 7.5, 100 mM NaCl, and 0.05% Tween-20) and washed with TBS-T prior to incubation with a rabbit polyclonal antibody against NOX4 (Abcam, Cambridge, UK) at room temperature for 2 h. ß-Actin (Santa Cruz Biotechnology, Santa Cruz, USA) served as marker for equal protein loading. After washing with TBS-T, HRP- (horseradish peroxidase-) conjugated anti-rabbit IgG antibody (New England Biolabs) was added. Immunoreactive proteins were visualized by chemiluminescence (Amersham Chemiluminescence Kit) and exposure to X-ray films (Agfa, Mortsel, Belgium). Densitometrical analysis was performed using the freely available image-processing software ImageJ 1.43 (http://rsb.info.nih.gov/ij/).
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9

Protein Expression Analysis Protocol

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Cells were harvested, washed with PBS, and lysed using RIPA buffer. Cell lysates were separated using SDS PAGE and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk, the membranes were incubated with the primary antibodies (1:1000) at 4 °C overnight, followed by incubation with the horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) for 1 h. Development was performed using Clarity Western ECL Substrate (Bio-Rad, Gladesville, Australia) and detected on X-ray films (AGFA, Greenville, SC, USA) or using ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, USA).
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10

Quantification of DENV mRNA Isoforms

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Cells were mock infected or infected (MOI ~1) with DENV WT and harvested 48 hours post-infection. Total RNA was extracted by using Trizol (Invitrogen). The isolated RNA was subjected to RT-PCR in presence of α-32P dCTP. The specific primers are listed in S1 Table. Radiolabelled PCR products were electrophoresed in 6% polyacrylamide native gels, which were subsequently dried and exposed to X-ray films (Agfa). The bands detected by autoradigraphy were excised from gel and the radioactivity was measured in a scintillation counter (Cerenkov method) to then calculate the ratio between the amplification products corresponding to different mRNA isoforms.
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