In order to isolate protein, spheroids were harvested with RIPA Lysis and Extraction buffer (Thermo Fisher Scientific) supplemented with cOmplete protease and PhosSTOP inhibitors (Roche). Aliquots of the protein homogenate were subjected to SDS-PAGE and Western blot analysis according to Karlgren et al.26 (link) with minor modifications. AmershamProtran membrane (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) was used and visualization was performed using the Super Signal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Waltham, MA USA). Primary antibodies for GSK3β (#9832 CellSignal) and Phospho-GSK-3β (#9336 CellSignal) were used.
Amersham protran membrane
Manufactured by GE Healthcare
Sourced in United Kingdom
Amersham Protran membrane is a nitrocellulose-based membrane used for protein and nucleic acid transfer and immobilization in western blotting, northern blotting, and other laboratory applications. It provides a high-binding capacity for biomolecules and is suitable for use with a variety of detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop inhibitor, by Roche (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Cl xposure film, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence ecl kit, by Cytiva (1 mentions)
Optiprep media, by Merck Group (1 mentions)
Mono poly resolving media, by MP Biomedicals (1 mentions)
Bradford protein assay reagent, by Bio-Rad (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Protease inhibitor tablet, by Roche (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Ab3114, by Santa Cruz Biotechnology (1 mentions)
Odyssey software, by LI COR (1 mentions)
P irf3, by Abcam (1 mentions)
H6908, by Abcam (1 mentions)
Secondary abs, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
F7425, by Merck Group (1 mentions)
5 protocols using amersham protran membrane
1
Protein Isolation and Western Blot Analysis
2
Infrared Western Blotting Analysis Protocol
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Infrared western blotting analyses were performed as previously described (22 (link)). In brief, cells were harvested by scraping and total protein was extracted by using lysis buffer (50 mM Tris–HCl, pH 7.4, 50 mM NaCl, 2 mM MgCl2, 1% SDS) supplemented with protease inhibitor cocktails (Sigma-Aldrich). Proteins were separated in a NuPAGE 4–12% Bis-Tris gel (Thermo Fisher Scientific) and transferred to an Amersham Protran membrane (GE Healthcare). Protein expression was determined by using specific primary and fluorescence-coupled secondary antibodies and an infrared Odyssey Scanner (LI-COR). Antibodies are summarized in Supplementary Table S9 .
3
Detailed Blood Collection and Protein Analysis Protocol
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Vacutainers for blood collection were purchased from BD (Wokingham, United Kingdom) (367869), Optiprep media (D1556), lithium chloride (203637), and sodium chloride (S7653) were from Sigma (Gillingham, United Kingdom), Mono-Poly resolving media from MP Biomedicals (Santa Ana, United States) (0916980). Protease inhibitor tablets were from Roche (11836170001) and Bradford protein assay reagent was from Biorad (Watford, United Kingdom) (500-0006). The nitrocellulose (Amersham Protran) membrane and Phosphocellulose P81 paper were from GE Healthcare Life Sciences (Little Chalfort, United Kingdom) (10600002), the Enhanced Chemiluminescence (ECL) Kit used was from Amersham (RPN2209) and CL-Xposure film from Thermo Scientific (Waltham, United States). The radioisotope, γ-32P-ATP was purchased from Perkin Elmer (Waltham, United States). All other chemicals were of the highest grade available. All antibodies used in this study are detailed in Table 2 .
4
Western Blot Analysis of Cellular Proteins
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Proteins in cell lysates were separated by electrophoresis and transferred to a nitrocellulose Amersham Protran membrane (GE Healthcare). The membrane was blocked for 1 hr in 5% milk in PBS containing 0.1% Tween-20 at 23°C. Then, the membrane was incubated with primary antibody (Ab) diluted in blocking buffer at 4°C overnight. Antibodies used were from the following sources: DNA-PKcs (NeoMarkers, MS-423-P1, diluted 1:1000), Ku70 (AbCam, ab3114, diluted 1:1000), Ku80 (Santa Cruz, sc-1484, 1:500), FLAG (Sigma-Aldrich, F7425, diluted 1:5000), HA (Sigma-Aldrich, H6908, diluted 1:1000), P-IRF3 (Abcam, ab76493, diluted 1:1500), and α-tubulin (Merck Millipore, 05-829, diluted 1:10000). Membranes were probed and visualized with LI-COR Biosciences secondary Abs and the Li-Cor Odyssey infrared imaging system according to the manufacturer’s instructions. A quantification of protein bands was performed, where indicated, by using Odyssey software (LI-COR Biosciences).
5
Western Blot Analysis of Machi3-1
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Nectars and the protein extracts were separated by stainfree 1D SDS-PAGE (Bio-Rad's Mini PROTEAN ® TGX Stain-Free™ Gels). For Western-blot assays, samples were separated by SDS-PAGE, blotted onto Amersham Protran membrane (GE Healthcare, 10600008) and hybridized with rabbit polyclonal antibody serum raised against Machi3-1. ECL Anti-Rabbit IgG Horseradish Peroxidase linked (GE Healthcare, NA934-1ML) secondary antibody was used for detection. Actin antibody (Anti-Actin Plant MerckA0480) was used for control. Chemiluminescent protein detections were conducted with ECL Western Blotting Substrate (Promega, W1001), according to the manufacturer's instructions. Western blots were scanned with ChemiDoc MP System and analyzed with ImageLab 5.0 software (Bio-Rad).
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