Fly heads were homogenized on ice in radioimmune precipitation buffer (RIPA, containing 50 mm Tris–HCl, 150 mm NaCl, 0.1% sodium deoxycholate [SDS],, 0.5% Na, 1% NP-40, [pH 8.0]) supplemented with protease inhibitor (Complete Protease inhibitor, Roche, Indianapolis, IN, USA). Homogenates were incubated on ice for 20 min and protein lysates were extracted by centrifugation (12000 rpm for 20 min at 4°C). Supernatants were used for immunoblotting. Protein concentrations were measured by Micro BCA assay kit (ThermoFisher Scientific; 23235). Western blotting was performed as described before (25 (link)). Briefly, 30 μg of protein was loaded on a 7.5% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. Membranes were blocked in 5% skimmed milk in PBS and probed with primary antibodies. Immunodetection was performed with specific secondary antibodies conjugated to horseradish peroxidase and the ECL-plus chemiluminescent detection system (Amersham Biosciences, Little Chalfont, UK) with bands quantified on a LAS-3000 Station (GE Healthcare, Chicago, IL, USA). The signal was normalized to the signal obtained from tubulin for quantification. Primary antibodies were used in a 1/500 dilution: rabbit anti-FUS (Bethyl Laboratories, A300-302A) and rabbit anti-tubulin (Cell signaling, #2125).
Ecl plus chemiluminescent detection system
Manufactured by Cytiva
Sourced in United Kingdom
The ECL Plus chemiluminescent detection system is a laboratory equipment designed for the detection and quantification of proteins in Western blot analysis. It utilizes a chemiluminescent reaction to produce a luminescent signal proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti tubulin, by Cell Signaling Technology (1 mentions)
Micro bca assay kit, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Mouse monoclonal anti β actin antibody clone ac 15, by Merck Group (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Hybond p, by Cytiva (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Gapdh, by GeneTex (1 mentions)
Hybond, by Cytiva (1 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Western blocking reagent, by Roche (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Mcl1 1kt, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
6 protocols using ecl plus chemiluminescent detection system
1
Fly Head Protein Extraction and Western Blot
2
Protein Extraction and Western Blot Analysis
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JURKAT or CCRF-CEM cells (3 × 106 per sample) were stimulated as described above and lysed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Non-idet P-40, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM Na3VO4, 1 mM NaF, and 20 μg/ml aprotin/leupeptin/PMSF). Lysates were centrifuged at 4°C, 13,000 × g for 30 min, and the supernatants were collected. Then, 30 μg of each sample was resolved by SDS-PAGE, and proteins were transferred to nitrocellulose membranes. The membranes were washed with TBST and blocked with 5% non-fat dry milk or 3% BSA and then incubated overnight at 4°C with each indicated primary Ab and HRP-conjugated secondary Ab. Signal was detected using ECL Plus chemiluminescent detection system (Amersham).
3
Quantitative Western Blot Analysis
4
Quantifying TBK1 Protein Levels
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Protein lysates from the TBK1 p.Thr79del carrier and control frontal cortex tissue were prepared for Western blot with 0.1% RIPA and sonicated on ice, cleared at 20,000 g for 15 min at 4°C and supernatants used for immunoblotting (Fig. 1 ). Protein concentrations were measured by a BCA assay (Pierce, Rockford, IL), and 30 μg of protein were separated on 4%–12% Nupage Bis–Tris gels (Invitrogen), electroblotted onto a PVDF membrane (Hybond P; Amersham Biosciences, GE Helthcare, Little Chalfont, UK), and probed with a monoclonal antibody against TBK1 (Abcam, Cambridge, MA; 1:1,000, 84 kDa). Immuno‐detection was performed with specific secondary antibodies conjugated to horseradish peroxidase and the ECL‐plus chemiluminescent detection system (Amersham Biosciences). TBK1 signal intensities were quantified against GAPDH (Genetex, Irvine, CA; 1:10,000, 37 kDa), using ImageQuantTL software (GE Healthcare Life Sciences, Little Chalfont, UK).
5
Protein Extraction and Immunoblotting from Aphids
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Fifteen aphids (from a mix of distinct nymphal and adult life stages) were pooled, washed with PBS, and gently crushed in cell lysis buffer (MCL1-1KT; Sigma-Aldrich) supplemented with protease inhibitor mixture (Roche). Aphid tissue lysate was incubated on ice for 15 min and then centrifuged (12,000 × g, 5 min, 4 °C). The supernatant was used for immunoblotting, and its protein concentration was determined using the Bio-Rad Protein assay (Bio-Rad Laboratories). Twenty micrograms of protein were separated on Bolt 4–12% Bis-Tris Plus gels (Thermo Fisher Scientific) using Bolt Mes SDS running buffer and were blotted onto a nitrocellulose membrane. Membranes were blocked with Western Blocking Reagent (Roche) and probed with primary antibodies. Immunodetection was performed with HRP-conjugated secondary antibodies and the ECL-Plus chemiluminescent detection system (Amersham Biosciences).
6
Pulmonary Protein Analysis in Lung Biopsies
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Lung biopsies available were peer-reviewed independently and blinded by a pathologist specialized in pulmonary pathology. Lung tissue of patient 3 was analyzed by Western blotting under reducing and denaturing conditions using sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by electroblotting and immunostaining for pro-SP-C (Merk Millipore, Darmstadt, Germany), ATF6, β-actin (abcam, Cambridge, UK), and cleaved caspase-3 (Cell Signaling, Gaithersburg, USA). Blotted membranes were developed with the ECL Plus chemiluminescent detection system (Amersham Biosciences, Amersham, UK). Immunohistochemistry was performed on lung tissue fixed in 4% formaldehyde on serial sections with the AP Fast Red kit (Zytochem Systems, Berlin, Germany) after antigen retrieval by microwaving in 10 mM sodium citrate buffer, pH 6.0. Hemalaun was used as counter-stain. Slides from patient 2 were also available for immunostaining for pro-SP-C and cleaved caspase-3, as described above. As controls, lung sections from 3 different organ donor lungs were used.
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