Wandering L3 larvae or adult female flies were used for protein isolation. Collected larvae or adult flies were lysed in I-RIPA protein lysis buffer (150mM NaCl, 50mM Tris-HCl pH 7.5, 1mM EDTA, 1% v/v Triton X-100, 1% w/v Na Deoxycholic Acid, 1xprotease inhibitor cocktail), protein extracts were resolved on a 4–20% w/v gradient polyacrylamide gel (Bio-Rad, Cat. No. 456–1093), transferred to Immobilon®-P membrane (Millipore, Cat. No. IPVH00010) and probed with rat anti-TRAL (1:2000, this study), rat anti-PATR145 (link) (1:2000) or mouse anti-a-tubulin (12G10, Developmental Studies Hybridoma Bank, 1:1000) antibodies. Subsequently, blots were washed extensively with 1×TBST (1×TBS, 0.1% v/v Tween-20) and incubated with anti-rat or -mouse conjugated HRP secondary antibodies. After extensive secondary washes with 1×TBST, blots were treated with ECL-detection reagent 1 and 2 (Thermo Scientific, Cat. No. 1859701 and 1859698) and finally exposed to chemiluminescence films (GE Healthcare, Cat. No. 28906839) and developed the signal.
Chemiluminescence film
Manufactured by GE Healthcare
Sourced in United Kingdom
Chemiluminescence films are a type of laboratory equipment used to detect and analyze chemiluminescent signals. These films are sensitive to light produced by certain chemical reactions and are often used in applications such as western blotting, immunoassays, and gene expression analysis. The films capture the light emitted from the chemiluminescent process, allowing researchers to visualize and quantify the results of their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p membrane, by Merck Group (3 mentions)
Gradient polyacrylamide gel, by Bio-Rad (3 mentions)
Ecl detection kit, by GE Healthcare (2 mentions)
Ecl detection reagent, by Thermo Fisher Scientific (2 mentions)
Ab290, by Abcam (2 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Image studio software, by LI COR (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Merck Group (1 mentions)
Mouse anti flag, by Merck Group (1 mentions)
Mini protean 3 system, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Pierce ecl detection reagent, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
10 protocols using chemiluminescence film
1
Protein Isolation and Western Blot Analysis
2
Comprehensive Western Blotting Protocol
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Standard procedures were followed for Western blotting. Briefly, proteins were extracted with cell lysis buffer supplemented with EDTA-free Mini-Complete protease inhibitors and a PhosSTOP phosphatase inhibitor cocktail (Roche). Protein lysates were clarified and separated using 4%–12% Bis-Tris NuPAGE gels (Thermo Fisher) and then transferred onto nitrocellulose membranes (GE Healthcare). Membranes were blocked with Odyssey blocking buffer (LI-COR) or 5% dried skimmed milk in TBS-T and incubated with primary antibodies overnight rocking at 4°C. To develop the blots, the membrane was incubated with appropriate IRDye 680LT-conjugated or IRDye 800CW-conjugated secondary antibodies for 1 h before detection using an Odyssey scanner (LI-COR). Images were analyzed using Image Studio software (LI-COR). Alternatively, membranes were incubated with appropriate HRP-linked secondary antibodies (CST) and developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and chemiluminescence films (GE Healthcare). ACTIN was used as a loading control performed on the same blot as the other panels shown in each figure. Primary and secondary antibodies used for Western blotting are listed in Supplemental Tables S6A and S7 .
3
Western Blot Analysis of Liver Proteins
4
Drosophila Protein Isolation and Western Blot
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Female Drosophila flies were used for protein isolation. Extract was prepared from either whole animals or separately from the intestines and remaining carcass tissue of dissected animals. Tissues were lysed in protein lysis buffer (25mM Tris-HCl pH 7.5, 150mM KCl, 5mM MgCl2, 1% NP-40, 0.5mM DTT, 1x Complete protease inhibitor cocktail (Sigma)), protein extracts were resolved on a 4-20% gradient polyacrylamide gel (Bio-Rad), transferred to Immobilon-P membrane (Millipore) and probed with rabbit anti-GFP (ab290, Abcam, 1:10,000) or mouse anti-a-tubulin (12G10, Developmental Studies Hybridoma Bank, 1:1000) antibodies. For IP verification, blots were stained with mouse anti-FLAG (F1804, Sigma, 1:3500) or mouse anti-FMRP (5A11, Developmental Studies Hybridoma Bank, 1:500). Subsequently, blots were washed extensively with 1xTBST (1xTBS, 0.1% Tween-20) and incubated with antirabbit or -mouse conjugated HRP secondary antibodies. After extensive secondary washes with 1xTBST, blots were treated with ECL-detection reagents (Thermo Scientific) and finally exposed to chemiluminescence films (GE Healthcare).
5
Protein Isolation from Adult Flies
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Adult female flies were used for protein isolation. Whole adult flies were lysed in I-RIPA protein lysis buffer (150mM NaCl, 50mM Tris-HCl pH 7.5, 1mM EDTA, 1% Triton X-100, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted January 4, 2022. ; https://doi.org/10.1101/2022.01.03.474858 doi: bioRxiv preprint 1% Na Deoxycholic Acid, 1xprotease inhibitor cocktail). Prepared protein extracts were resolved on a 4-20% gradient polyacrylamide gel (Bio-Rad, Cat. No. 456-1093) , transferred to Immobilon ® -P membrane (Millipore, Cat. No. IPVH00010) and probed with rat anti-Swm (this study,1:100), rabbit anti-GFP (ab290, Abcam, 1:10,000) or mouse anti-a-tubulin (12G10, Developmental Studies Hybridoma Bank, 1:1000) antibodies. After washing with 1xTBST (1xTBS, 0.1% Tween-20) blots were incubated with anti-rat, -rabbit, -or -mouse conjugated HRP secondary antibodies. Subsequently, blots were washed with 1xTBST, treated with ECL-detection reagents (Thermo Scientific, Cat. No. 1859701 and 1859698) and finally exposed to chemiluminescence films (GE Healthcare, Cat. No. 28906839) .
The copyright holder for this preprint this version posted January 4, 2022. ; https://doi.org/10.1101/2022.01.03.474858 doi: bioRxiv preprint 1% Na Deoxycholic Acid, 1xprotease inhibitor cocktail). Prepared protein extracts were resolved on a 4-20% gradient polyacrylamide gel (Bio-Rad, Cat. No. 456-1093) , transferred to Immobilon ® -P membrane (Millipore, Cat. No. IPVH00010) and probed with rat anti-Swm (this study,1:100), rabbit anti-GFP (ab290, Abcam, 1:10,000) or mouse anti-a-tubulin (12G10, Developmental Studies Hybridoma Bank, 1:1000) antibodies. After washing with 1xTBST (1xTBS, 0.1% Tween-20) blots were incubated with anti-rat, -rabbit, -or -mouse conjugated HRP secondary antibodies. Subsequently, blots were washed with 1xTBST, treated with ECL-detection reagents (Thermo Scientific, Cat. No. 1859701 and 1859698) and finally exposed to chemiluminescence films (GE Healthcare, Cat. No. 28906839) .
6
Biotin-PAA Synthesis and Protein Interactions
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The bespoke synthesis of N-Biotinyl p-aminophenyl arsinic acid (Biotin-PAA) was commissioned to SynInnova Laboratories Inc. Recombinant protein was treated under control conditions, with H2O2 (100 or 500 μM), 100 μM DTT or β-mercaptoethanol (5% v/v) for 15 min at room temperature. Protein samples were then re-suspended with an equal volume of 2x non-reducing SDS sample buffer. Protein samples were loaded and separated by SDS-PAGE using Mini-protean 3 system (Bio-Rad) and transferred to PVDF membranes (Bio-Rad). Membranes were blocked for 1 h at RT in Dilution Buffer from Pierce™ Far-Western Biotinylated Protein:Protein Interaction Kit (Thermo Fisher). Biotin-PAA was diluted to 0.4 mM in Dilution Buffer and incubated with the membrane for 1 h at RT. The membrane was then washed 6 times with PBS-T and subsequently incubated with Streptavidin-HRP at 1:10,000. Protein bands were visualized on chemiluminescence film (GE Healthcare) following incubation of the membranes with ECL Western Blotting Detection Reagents (Pierce).
7
Western Blot Analysis of IFIT1 Protein
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MEFs were induced for 72 h as described above and then lysed in radio immunoprecipitation assay buffer with protease and phosphatase inhibitors (2 (link)). Cell wall debris was cleared by centrifugation, and 20 ng of protein was electrophoresed on 5%/10% SDS polyacrylamide discontinuous gels. Gels were transferred to a polyvinylidene difluoride (PVDF) immunoblotting membrane by semidry transfer and blocked with 5% nonfat milk–Tris-buffered saline–0.1% Tween 20 (TBS-T) for 1 h. Anti-FLAG M2-peroxidase conjugate (1:2,000) was applied for 2 h at room temperature, or rabbit anti-mIfit1 (1:1,000) was applied overnight at 4°C with agitation. Secondary detection of IFIT1 was performed by thoroughly washing in TBS-T followed by goat anti-rabbit horseradish peroxidase (HRP) conjugate (1:2,000) for 1 h at room temperature. Blots were thoroughly washed prior to detection with Pierce ECL detection reagent (Thermo Scientific) and exposure to chemiluminescence film (GE Healthcare).
8
Recombinant APTX Protein Expression
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hAPTXcat WT and variants were grown in 3 ml LB medium containing ampicillin (100 μg/ml) and chloramphenicol (34 ng/ml) at 37°C. Cell cultures were induced with 100 μM IPTG when A600 = 1. Protein expression was carried out at 16°C overnight. Cell density was normalized to A600 = 1. Cell pellet from 1 ml cell culture was suspended and lysed in 200 μl (50 mM Tris, pH 8.0, 100 mM NaCl, 5 mM EDTA, 0.25 mg/ml lysozyme, 10 μg/ml DNaseI, 5 mM MgCl2) at 4° for 1 h. Cell lysate was fractionated by centrifugation (30,000 g, 10 min). Whole cell lysates (5 μl) and soluble fractions (5 μl) were boiled in 2× SDS sample buffer (5 μl) for 10 min, followed by centrifugation (30,000 g, 10 min). Western blotting for his‐tagged hAPTXcat WT and variant proteins in whole cell lysate and soluble fractions used HRP mouse anti‐His6 antibodies (1:5,000) (BD Pharmingen), followed by HRP‐conjugated rabbit anti‐mouse secondary antibodies (1:5,000). Membranes were treated by ECL detection kit (GE Healthcare) and then exposed to chemiluminescence film (GE Healthcare).
9
Protein Extraction and Western Blot Analysis of Sciatic Nerve Tissues
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Sciatic nerve tissues were homogenized in RIPA lysis buffer plus protease inhibitor cocktail (Roche). Lysates were keep rotating for 90 min at 4 °C and then spun at 13,000 rpm for 15 min. The protein concentration of the supernatant was determined using a BCA Protein assay kit (Thermo Scientific). Proteins (15 μg) were loaded in 10% Mini‐PROTEAN® TGX™ Precast Gels (Biorad) and transferred to a Nitrocellulose membrane (Biorad), blocked in 10% milk in PBS 0.1% Tween. Primary antibody used were αGt MME (Life Technologies; 1:1,000) and αMs GAPDH (Abcam; 1:500). Secondary antibodies were anti‐goat (Abcam; 1:20,000) and anti‐mouse (GE Healthcare; 1:10,000) IgG horseradish peroxidase linked. ECL prime detection kit (GE Healthcare) was used to visualise immunoreactive bands on chemiluminescence film (GE Healthcare). Quantification was done using ImageJ software and expression was normalised against GAPDH loading control.
10
Western Blot Protein Analysis
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Bacterial lysates, protein fractions from differential velocity ultracentrifugation and fast protein liquid chromatography with size exclusion chromatography (FPLC-SEC), and molecular weight standards were mixed with 2X sample buffer and heated at 100°C for 5 min. Samples were vortexed for 10 min prior to loading onto a discontinuous polyacrylamide gel with 5% stacking and 9% separating gels, and submerged in 1X Tris/SDS running buffer (Bio-basic Ltd.).
Proteins were separated by applying 130 Volts for 2 hours. Some gels were stained with Bio- WesternC Chemiluminescent solution (Bio-RAD) or 1 ml of ECL plus Western blotting dection system (GE Healthcare) for 5 min as described by the manufacturer. Blots were then wrapped in Saran Wrap and exposed to chemiluminescence film (GE Healthcare Ltd.) in a dark room for 30 sec to 3 min. The autoradiography film was developed using GBX developer and replenisher (Kodak, Rochester, NY, USA), and fixed using GBX fixer and replenisher (Kodak, Rochester, NY, USA).
Proteins were separated by applying 130 Volts for 2 hours. Some gels were stained with Bio- WesternC Chemiluminescent solution (Bio-RAD) or 1 ml of ECL plus Western blotting dection system (GE Healthcare) for 5 min as described by the manufacturer. Blots were then wrapped in Saran Wrap and exposed to chemiluminescence film (GE Healthcare Ltd.) in a dark room for 30 sec to 3 min. The autoradiography film was developed using GBX developer and replenisher (Kodak, Rochester, NY, USA), and fixed using GBX fixer and replenisher (Kodak, Rochester, NY, USA).
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