YD-38 cells were placed into 6-well plates at a density of 1×104 cells/ml and cultured, then treated with cisplatin (8 μM) with or without β-elemene (40 μg/ml) for 48 h. Cells were lysed and then total proteins were separated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and then the membrane was blocked for 1 h at room temperature. The membranes were incubated overnight with primary antibodies (STAT3, p-STAT3, JAK2, p-JAK2, caspase-3, Bcl-2, and Bax) at 1: 1000 dilution at 4°C. The membranes were then incubated for 2 h with horseradish peroxidase-labeled secondary antibodies (1: 5,000; affinity purified goat anti-mouse IgG and goat anti-rabbit IgG, Santa Cruz Biotech) at room temperature. The immunostained protein bands were developed using the enhanced chemiluminescence system (Immun-Star™AP Chemiluminescence kit; Bio-Rad Laboratories) and X-ray films (Santa Cruz Biotechnology Inc.). The blots were analyzed using Quantity One software, version 4.6 (Bio-Rad Laboratories).
X ray film
Manufactured by Santa Cruz Biotechnology
Sourced in Germany, United States
X-ray film is a photographic medium used to capture and record images produced by X-rays. It functions by exposing the film to X-ray radiation, which causes chemical changes in the film's emulsion layer, resulting in a latent image. The film can then be processed and developed to produce a visible image that can be used for various medical and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Luminol reagent, by Santa Cruz Biotechnology (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Precast polyacrylamide gel, by Bio-Rad (3 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Immun star ap chemiluminescence kit, by Bio-Rad (1 mentions)
Quantity one software version 4, by Bio-Rad (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Nfat1, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Ecl reagent, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence system, by Santa Cruz Biotechnology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Image lab v4, by Bio-Rad (1 mentions)
Ecl reagent solution, by GE Healthcare (1 mentions)
Epiquik nuclear extraction kit, by Epigentek (1 mentions)
Histone extraction kit, by Abcam (1 mentions)
12 protocols using x ray film
1
Cisplatin and β-elemene Regulation of STAT3 and Apoptosis
2
OVA-Induced BMDC Activation and NF-κB Signaling
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BMDCs were seeded onto a 100 mm Petri
dish at a density of 1 × 106 cells per well and incubated
overnight. The cells were then treated with OVA (10 μg/mL) or
OVA (10 μg/mL) plus AnCHNPs (5 μg/mL). After incubation
for 24 h, cells were harvested and lysed with a RIPA buffer supplemented
with 1× proteinase inhibitor cocktail (Amresco). Protein concentration
was determined using a bicinchoninic acid (BCA) protein assay (Thermo
Fisher Scientific). Protein lysates were loaded onto 10% SDS-PAGE
and transferred to a PVDF membrane. Nonspecific binding to the membrane
was blocked by incubation with 5% nonfat milk at room temperature
for 1 h. The membrane was incubated with primary antibodies at 4 °C
overnight at dilutions specified by the manufacturer. This is followed
by incubation with secondary antibodies for 1 h at room temperature
and then treatment with ECL reagents (Thermo Fisher Scientific). The
membrane was then exposed to X-ray films (Santa Cruz). All the imaging
results were analyzed by ImageJ. The antibodies used are NFAT1 (Cell
Signaling Cat # 4389S); phospho-IκBα, phospho-NF-κB
p65 (Cell Signaling Cat # 9936T); and GAPDH (Cell Signaling Cat #
5174S).
dish at a density of 1 × 106 cells per well and incubated
overnight. The cells were then treated with OVA (10 μg/mL) or
OVA (10 μg/mL) plus AnCHNPs (5 μg/mL). After incubation
for 24 h, cells were harvested and lysed with a RIPA buffer supplemented
with 1× proteinase inhibitor cocktail (Amresco). Protein concentration
was determined using a bicinchoninic acid (BCA) protein assay (Thermo
Fisher Scientific). Protein lysates were loaded onto 10% SDS-PAGE
and transferred to a PVDF membrane. Nonspecific binding to the membrane
was blocked by incubation with 5% nonfat milk at room temperature
for 1 h. The membrane was incubated with primary antibodies at 4 °C
overnight at dilutions specified by the manufacturer. This is followed
by incubation with secondary antibodies for 1 h at room temperature
and then treatment with ECL reagents (Thermo Fisher Scientific). The
membrane was then exposed to X-ray films (Santa Cruz). All the imaging
results were analyzed by ImageJ. The antibodies used are NFAT1 (Cell
Signaling Cat # 4389S); phospho-IκBα, phospho-NF-κB
p65 (Cell Signaling Cat # 9936T); and GAPDH (Cell Signaling Cat #
5174S).
3
Co-immunoprecipitation and Western Blot Analysis
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Co-imunoprecipitation was performed using Thermo-Fischer Pierce Co-immunoprecipitation Kit as per manufacturer’ protocol. The beads were boiled in 2x lysis buffer to release the immuno-complex. The samples were resolved on SDS-PAGE gel were transferred onto the nitro-cellulose membrane (MDI). The blots were blocked with 5% blocking at 37°C for 1 h followed by incubation with primary antibody overnight at 4°C. Subsequently, the blots were washed thrice for 5 minutes each with 1x PBST (Phosphate-buffered saline with 1% Triton-100) and were then incubated with secondary antibody for 2 h at 37°C. The blots were then washed thrice for 5 min each with 1x PBST. The blots were developed on X-ray films (Amersham or Kodak) after incubation with ECL reagent (Immunocruz, Santa Cruz biotechnology).
4
Protein Expression Analysis in Cells
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Following treatment, cells were lysed with lysis buffer and equal amounts of protein were electrophoresed using 10% sodium dodecyl sulfate-polyacrylamide gel, then transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The membranes were incubated with the following primary antibodies: Rabbit anti-rat PI3 Kinase p110α, AKT antibody, Phospho-Akt Antibody, Bcl-2 Antibody, Bax Antibody, Caspase-3, and mouse anti-rat GAPDH monoclonal antibody (1: 1,000). We then incubated them with the HRP conjugated goat anti-rabbit IgG secondary antibody (1: 10000). The membranes were visualized using an enhanced chemiluminescence system and X-ray films (Santa Cruz Biotechnology Inc.).
5
Apoptosis Signaling Pathway Analysis
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Four groups were divided as the control group, Cisplatin group and PSI + Cisplatin group. The concentration of DMSO in the control group was 0.25% (v/v). Cells were treated with PSI (0.3 μg/ml) and Cisplatin (16 μM), Following treatment, cells were lysed with lysis buffer containing 50 mM Tris-HCl (pH 8.0) and 150 mM 1% TritonX-100 (Sigma-Aldrich). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to nitrocellulose membranes (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The membranes were incubated overnight at 4°C with primary antibodies (rabbit anti-rat Bcl-2, Bax, caspase-3 and p21/Waf1/Cip1 monoclonal antibodies and mouse anti-rat GAPDH monoclonal antibody (1: 1,000)), and then washed three times with Tris-buffered saline supplemented with Tween-20, prior to being incubated for 2 h at room temperature with the HRP-conjugated goat anti-rabbit IgG secondary antibody (1: 10,000). The membranes were visualized using an enhanced chemiluminescence system and X-ray films (Santa Cruz Biotechnology Inc.).
6
Nuclear Protein Extraction and Immunoblotting
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Total protein extracts were obtained lysing cultured cells with ice-cold RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1% Nonidet P-40) supplemented with protease and phosphatase inhibitors (Sigma). Cellular extracts optimized to preserve DNMT enzymatic activity assay were obtained through the EpiQuik™ Nuclear Extraction Kit (EpiGentek Group Inc, NY, United States) following the manufacturer’s instructions. Purification of total histones was obtained according to the protocol provided by the Histone Extraction Kit (Abcam). Protein concentration was determined through the Bradford method, and samples were subjected to 10–12% SDS-PAGE. Gels were electroblotted into nitrocellulose membranes that were probed with the primary antibodies described above. Membranes were incubated with enhanced chemiluminescence (ECL) reagent solution (GE Healthcare, Hilden, Germany) and exposed to X-ray film (Santa Cruz). Immunoreactive band density was quantified using ImageLab v4.0 analysis software (Bio-Rad, Hercules, CA, United States).
7
SARS-CoV-2 ORF7a and ORF7b Expression
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293T-hACE2 cells were transfected using Lipofectamine 3000 (Invitrogen, L3000–015) with plasmids encoding for ORF7aWT, ORF7aΔ115 or control pEGFP-N1 plasmid. To detect ORF7b expression cells were infected with ORF7aWT or ORF7aΔ115 SARS-CoV-2 strains at MOI = 0.05. After 24 hours, cells were washed two times with PBS and lysed in RIPA buffer (150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.2% SDS, 50 mM Tris, pH 8.0) at 4C for 30 min. The lysates were clarified by centrifugation (10,000 g, 20 min) and stored at −80C. For Western blot lysates were mixed with 6xLaemmli SDS-PAGE buffer and heated at 98C for 5 min, resolved in 12% SDS-PAGE gel and transferred onto a PVDF (ORF7b) or nitrocellulose (ORF7a) membrane using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, #1703930). Membranes were blocked and probed with indicated antibodies. The proteins were visualized using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, #32106) and exposed to X-Ray film (sc-201696, Santa Cruz Biotech).
8
Western Blot Quantification Protocol
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Forty μg of heat-denatured proteins were loaded on 4–15% precast polyacrylamide gel (Bio-Rad, USA). The proteins were then transferred to PVDF membranes (Bio-Rad, USA.), which were blocked with 5% non-fat milk solutions for 1 hour at room temperature. The target proteins were then detected by the primary antibody at 4 °C overnight, washed with 0.1% Tween-PBS and incubated with appropriate secondary antibody for 2 hours. The membranes were then washed and the target proteins were detected with luminol reagent and X-ray film (Santa Cruz). Quantification of the target protein was done using Photoshop (Adobe,USA). In short, the background of the target protein and β-actin were subtracted. Then, the relative expression of the target protein was normalized to β-actin and compared to that of the control group in each experiment.
9
Western Blot Analysis of HIF-1α and BIRC3
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40 μg of heat-denatured proteins were loaded on 4–15% precast polyacrylamide gel (Bio-Rad). The proteins were then transferred to PVDF membranes (Bio-Rad), which were blocked with 5% non-fat milk solutions for 1 hour at room temperature. The target proteins were then detected by the primary antibody at 4 °C overnight, washed with 0.1% Tween-PBS and incubated with appropriate secondary antibody for 2 hours. The membranes were then washed and the target proteins were detected with luminol reagent and X-ray film (Santa Cruz). Quantification of the target protein was done using Adobe Photoshop. In brief, the background of the target protein and β-actin were subtracted. Then, the relative expression of the target protein was normalized to β-actin and compared to that of the control group in each experiment. Anti-human HIF-1α antibody was from Novus-bio. Rabbit anti-human BIRC3, goat anti-Rabbit IgG-HRP and anti-β-actin IgG-HRP were obtained from Santa Cruz Biotech.
10
Western Blot Analysis of Proteins
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Heat-denatured proteins (50–100 µg) were loaded on 4–15% precast polyacrylamide gel (Bio-Rad, Hercules, CA, USA) along with molecular weight marker. The gel was run for 1–2 h at 120 V and then transferred to PVDF membranes (Bio-Rad) by semi-dry system. Membranes were blocked with 5% non-fat milk, 0.1% TBS-T (Tween) solution for 1 h at room temperature. The target proteins were then detected by the primary antibody at 4 °C overnight, washed with TBS-T (5 min each) and incubated with appropriate secondary antibody at room temperature for 1 h. The membrane was then washed three times with TBS-T, 5 min each. The target proteins were detected with luminol reagent and X-ray film (Santa Cruz Biotech, Dallas, TX, USA).
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