Cells were collected by scraping or trypsinization, washed in ice-cold phosphate-buffered saline (PBS), and lysed in lysis buffer complemented with protease inhibitor cocktails (MilliporeSigma) and phosphatase inhibitor cocktails (Thermo Fisher) for 40 min on ice. NETN300 (300 mM NaCl, 50 mM Tris pH 7.5, 0.2 mM EDTA, 1% NP40) was used for whole-cell lysis and NETN150 (150 mM NaCl, 50 mM Tris pH 7.5, 0.2 mM EDTA, 1% NP40) was used for co-immunoprecipitation (IP) assays. The lysates were then cleared by centrifugation at 15,000 rpm at 4 °C for 10 min. The protein concentration of supernatants was measured using the Bradford assay so that 20–30 μg of protein was loaded onto an SDS-PAGE gel and transferred to PVDF membranes (MilliporeSigma). Membranes were incubated with the indicated primary and secondary antibodies, and HRP signal was detected by enhanced chemiluminescence (ECL) Western blotting substrates (Thermo Fisher) using Hyblot CL autoradiography film (Thomas Scientific) or the iBright imager (Thermo Fisher). Antibody information can be found in Table S3 .
Hyblot cl autoradiography film
Manufactured by Thomas Scientific
Sourced in United States
HyBlot CL is an autoradiography film used for the visualization and detection of radioactively labeled biomolecules, such as nucleic acids and proteins, after separation by gel electrophoresis or other techniques. The film is sensitive to the radioactive emissions from the labeled samples, allowing for the capture of the resulting signal.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (9 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (6 mentions)
Immobilon p membrane, by Merck Group (5 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Pvdf membrane, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (3 mentions)
Bca assay, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Ibright imager, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor, by Cell Signaling Technology (2 mentions)
Lysis buffer, by Cell Signaling Technology (2 mentions)
Whatman, by Cytiva (2 mentions)
66 protocols using hyblot cl autoradiography film
1
Western Blot Analysis of Protein Lysates
2
Immunoblot Analysis of Cellular Proteins
3
Phospho-Receptor Tyrosine Kinase Array
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Cells were seeded in 3D as described above, lysed, and protein concentration in each sample was measured by the Micro BCA protein assay kit (Thermo Scientific #23235). Three hundred milligrams of protein were analyzed using the Human Phospho-Receptor Tyrosine Kinase Array Kit (R&D Systems, ARY001B) according to the manufacturer’s protocol. Array membranes were incubated with chemiluminescence detection solution, exposed to HyBlot CL autoradiography film (Thomas Scientific #E3012), and developed using a Kodak X-Omat processor (Kodak).
4
Protein Extraction and Western Blotting
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Protein samples were collected and lysed using radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (Cell Signaling Technology). Lysates were quantified by Bradford assay (Bio-Rad), and 10 μg of extracted protein was separated using Novex SDS–polyacrylamide gel electrophoresis reagents and transferred to nitrocellulose membranes (Life Technologies). Membranes were blocked in 5% milk protein and incubated with primary antibodies (table S3) overnight followed by secondary horseradish peroxidase–linked goat anti-rabbit and anti-mouse (Cell Signaling Technology) antibodies (1:1000) according to the manufacturers’ instructions. Antibody-bound membranes were incubated with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific) and developed using HyBlot CL autoradiography film (Thomas Scientific). The antibodies that used immunoblotting are listed in table S3.
5
Western Blot Analysis of Protein Expression
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Islets were isolated and hand-picked in the manner described above. They were washed in PBS before being lysed in ice-cold RIPA buffer with protease inhibitors, sonicated (Qsonica, Newtown, CT), centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatant was collected. Protein lysates were resolved by SDS-PAGE and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were probed overnight at 4 °C using the primary antibodies (Electronic Supplemental Table 2 ) indicated followed by secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. Immunoblots were developed with Super Signal West Femto HRP substrate (ThermoFisher Scientific), exposed to HyBlot CL autoradiography film (Thomas Scientific, Swedesboro, NJ) and were imaged using an SRX-101 film processor (Konica Minolta Medical Imaging., Wayne, NJ). Protein bands were quantified using ImageJ software and normalized against that of GAPDH.
6
Western Blot Protein Analysis
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Protein samples were collected and lysed using a radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (Cell Signaling Technology). Lysates were quantified by Bradford assay (Bio-Rad), and 10 μg of extracted protein was separated using Novex SDS–polyacrylamide gel electrophoresis reagents and transferred to nitrocellulose membranes (Bio-Rad Laboratories). Membranes were blocked in 5% milk protein and incubated with primary antibodies overnight, followed by secondary horseradish peroxidase–linked goat anti-rabbit and anti-mouse antibodies (1:1000) according to the manufacturer’s instructions. Antibody-bound membranes were incubated with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific) and developed using HyBlot CL autoradiography film (Thomas Scientific). The antibodies used for immunoblotting are listed in Supplemental data Table S1 .
7
Phospho-Kinase Profiling of Riluzole's Effect
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On Day 0, cells were seeded in a 6-well plate at 500 000 cells/well (SUM44, LCCTam). For MM134 and MM134 LTED, 1.5 million cells were seeded in 10 cm culture dishes. Forty-eight hours later, on Day 2, cells were treated with either 10μM riluzole or DMSO solvent as a control for 48 hours. On Day 4, cells were collected in 100 μL lysis buffer/well before determining total protein concentration by bicinchoninic acid (BCA) assay (#23225, ThermoFisher). According to the manufacturer's instructions, 500 micrograms of whole cell lysate were then assayed using the Human Phospho-Kinase Proteome ProfilerTM Array (#ARY003B [SUM44/LCCTam] and ARY003C [MM134/MM134 LTED], Bio-Techne). Array membranes were visualized using chemiluminescence detected by HyBlot CL autoradiography film (#E3018, Thomas Scientific, Swedesboro, NJ), then films were scanned and analyzed using FIJI [36 (link)]. A ratio of background-corrected intensity values for targets (phospho-kinase spots) to references (control spots) was created for each condition (DMSO and riluzole) within each cell line. Data are presented as the mean of the riluzole to DMSO ratio for 2 technical replicates from a single experiment for each cell line.
8
Western Blot Analysis of T Reg Proteins
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T regs were cultured as described previously ( 14). Protein was extracted using 2 × 10 6 T regs as one sample. T regs were broken up using lysis buffer (Cell Signaling Technology, Danvers, MA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% polyacrylamide gels followed by electroblotting onto nitrocellulose membranes (Whatman, Florham Park, NJ). The latter were incubated for 60 min in a solution containing 5% skimmed milk powder in PBS with 0.1% Tween (PBS-T). Subsequently, nitrocellulose membranes were incubated overnight at 4°C with the suitable primary antibody. Primary antibodies were rabbit monoclonal antibodies from Abcam: phosphorylated (p)-CREB (E113), CREB (E306), Foxp3 (EPR22102-37), and β-actin (ab8227). The next day, nitrocellulose membranes were rinsed with PBS-T and exposed to the suitable peroxidase secondary antibody for 1 h at ambient temperature. Protein bands were identified using an improved chemiluminescent horseradish peroxidase substrate (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Fisher Scientific, Waltham, MA). Protein bands were visualized by exposing them to HyBlot CL Autoradiography Film (Thomas Scientific, Swedesboro, NJ) and processing them using an x-ray film processor (Ecomax; Protech Medical Systems, Oberstenfeld, Germany).
9
Whole Cell Lysis and Co-Immunoprecipitation
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Cells were collected by scraping or trypsinization, washed in ice-cold phosphate buffered saline (PBS), and lysed in lysis buffer complemented with protease inhibitor cocktails (MilliporeSigma) and phosphatase inhibitor cocktails (Thermo Fisher) for 40 min on ice. NETN300 (300 mM NaCl, 50 mM Tris pH 7.5, 0.2 mM EDTA, 1% NP40) was used for whole cell lysis and NETN150 (150 mM NaCl, 50 mM Tris pH 7.5, 0.2 mM EDTA, 1% NP40) was used for coimmunoprecipitation (IP) assays. The lysates were then cleared by centrifugation at 15,000 rpm at 4 °C for 10 min. Protein concentration of supernatants was measured using the Bradford assay so that 20-30 µg of protein was loaded onto an SDS-PAGE gel and transferred to PVDF membranes (MilliporeSigma). Membranes were incubated with the indicated primary and secondary antibodies, and HRP signal was detected by enhanced chemiluminescence (ECL) Western blotting substrates (Thermo Fisher) using Hyblot CL autoradiography film (Thomas Scientific) or the iBright imager (Thermo Fisher). Antibody information can be found on Table S3.
10
Western Blot Protein Analysis
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For western blot analysis, equal amounts of proteins were separated using Novex WedgeWell 4-20%, Tris-Glycine gels (Invitrogen, Thermo Fisher Scientific). Proteins were transferred onto PVDF membranes (Thermo Fisher). Membranes were blocked with a solution of 5% non-fat dry milk (Bio-Rad Laboratories) in Tris Buffered Saline -0.5% Tween-20 (TBS-T), then incubated with primary antibody in a solution of 5% bovine serum albumin (BSA) (Bio-Rad Laboratories) in TBS-T overnight at 4 °C. Membranes were then washed in TBS-T solution, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (anti-mouse, #7076, or anti-rabbit, #7074, Cell Signalling Technology, Inc.) in 5% non-fat dry milk in TBS-T, for 1 h at room temperature. After additional washes, membranes were incubated with a chemiluminescent detection reagent (Western Lightning Plus, Chemiluminescent Substrate, PerkinElmer Inc.), and imaged on HyBlot CL Autoradiography Film (Thomas Scientific), and the film was developed using developer and fixer solutions (Merry X-Ray Imaging, Inc.). The following primary antibodies were employed: HSP90 (#4877), IκBα (#4812), NAMPT (#6122), β-actin (#3700) and Flotillin-2 (#3436), all from Cell Signaling Technology, Inc. Quantification of Western blots was performed using the ImageJ software (103) .
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