Skeletal muscle and heart were snap-frozen in liquid nitrogen and stored at −80 °C until use. Skeletal muscle and heart were thawed and homogenized in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris-HCl, pH 8.0) supplemented with a protease and phosphatase inhibitor cocktail by using a bead homogenizer (Precellys 24 homogenizer, Bertin Instruments, Paris, France). Protein concentrations were determined using a BCA assay. Homogenates (60 µg) were separated by SDS-PAGE using the Bio-Rad Stain Free gel system to enable total protein visualization and quantitation. Proteins on PAGE gels were transferred to PVDF membranes (Immobilon-FL, EMD Millipore), blocked in EveryBlot blocking buffer (Bio-Rad), and incubated with primary antibodies overnight at 4 °C. After washing in PBS containing 0.2% Tween-20 (PBST), membranes were incubated with fluorophore-tagged Starbright secondary antibodies (Bio-Rad) for 1 h at room temperature. After washing in PBST, blots were imaged on a Chemidoc MP system (Bio-Rad). Total protein using stain-free gels (Bio-Rad), after transfer of the proteins to the PVDF membrane (Immobilon®-FL, Millipore), the PVDF membrane was imaged by using a ChemiDocTM MP imaging system (Bio-Rad). All antibodies used are provided in Supplementary Table 3 .
Immobilon fl
Manufactured by Merck Group
Sourced in United States, Germany, Ireland
Immobilon-FL is a membrane-based system designed for the immobilization and detection of biomolecules, such as proteins and nucleic acids. It provides a versatile platform for various analytical and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (40 mentions)
Odyssey blocking buffer, by LI COR (31 mentions)
Odyssey imaging system, by LI COR (13 mentions)
Immobilon p, by Merck Group (10 mentions)
Image studio software, by LI COR (10 mentions)
Odyssey clx, by LI COR (10 mentions)
Odyssey scanner, by LI COR (9 mentions)
Irdye 680rd, by LI COR (9 mentions)
Bca assay kit, by Thermo Fisher Scientific (8 mentions)
Irdye 800cw, by LI COR (8 mentions)
Odyssey infrared scanning system, by LI COR (7 mentions)
Odyssey clx imager, by LI COR (7 mentions)
Image studio lite, by LI COR (7 mentions)
Nupage, by Thermo Fisher Scientific (6 mentions)
Infrared dyed secondary antibodies, by LI COR (6 mentions)
Odyssey clx imaging system, by LI COR (5 mentions)
Complete protease inhibitor cocktail, by Roche (5 mentions)
Odyssey infrared imager, by LI COR (5 mentions)
Ripa buffer, by Cell Signaling Technology (5 mentions)
Pharos fx plus molecular imager, by Bio-Rad (5 mentions)
241 protocols using immobilon fl
1
Stain-free Western Blot Quantification
2
Western Blot Analysis of Cas9 and Actin
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For western blotting, samples were prepared in a 4× Laemmli buffer and run on either 8% or 12% Tris-tricine gels for electrophoresis depending upon the molecular weight range being detected using this method. Following this, gels were electro-blotted on the polyvinylidene fluoride (PVDF) membrane (Immobilon-FL; Merck-Millipore). Blocking of membranes was carried out by incubation with the Odyssey Blocking Buffer (LI-COR Biosciences) for 20–30 min, followed by both primary and secondary antibody incubations for 1 h each at room temperature, each of which was followed by three washes of 5 min each.
Detection of Cas9 and β-actin was carried out using mouse anti-Cas9 antibody (Clontech) and rabbit anti-beta actin antibody (LI-COR Biosciences), respectively. Secondary antibodies used were either IR dye 680 goat anti-mouse or IR dye 800 goat anti-rabbit (LI-COR Biosciences).
For immunoblotting of proteins incorporated into virus-like particles, the particle-containing supernatant was concentrated on a sucrose cushion by ultracentrifugation at 100,000 × g for 2 h at 4°C. The pellet obtained was resuspended in the 2× Laemmli buffer containing 2X-PIC (protease inhibitor cocktail) and 50 mM TCEP (Tris[2-carboxyethyl]phosphine hydrochloride).
Detection of Cas9 and β-actin was carried out using mouse anti-Cas9 antibody (Clontech) and rabbit anti-beta actin antibody (LI-COR Biosciences), respectively. Secondary antibodies used were either IR dye 680 goat anti-mouse or IR dye 800 goat anti-rabbit (LI-COR Biosciences).
For immunoblotting of proteins incorporated into virus-like particles, the particle-containing supernatant was concentrated on a sucrose cushion by ultracentrifugation at 100,000 × g for 2 h at 4°C. The pellet obtained was resuspended in the 2× Laemmli buffer containing 2X-PIC (protease inhibitor cocktail) and 50 mM TCEP (Tris[2-carboxyethyl]phosphine hydrochloride).
3
Western Blot Analysis of nsP3 Protein
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Cells were transfected or infected as above, then harvested in 100 µL PLB. Protein concentration of samples was calculated using a Bradford assay. For nsP3 blots, cell lysate containing 30 µg of total protein in Laemmli buffer were incubated at 95 °C for 5 min prior to loading on a 10% SDS-PAGE gel. Proteins were then transferred from gels onto membranes (Immobilon FL, Merck Millipore) using a semi-dry blotter for 1 h at 15 V. Membranes were blocked using blocking buffer (LICOR) for 20 min at room temperature prior to incubation with primary antibodies; rabbit anti-nsP344 (link) or mouse anti-actin (Sigma) prepared in TBS. Membranes were incubated with primary antibody rocking overnight at 4 °C. Membranes were washed in TBS then secondary antibodies (LICOR) added for 1 h at rt. Membranes were then washed in TBS + 0.1% Tween, then dH2O prior to drying in the dark. Blots were imaged using the LICOR Odyssey scanner.
4
Western Blot Analysis of Stem Cell Markers
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Under normal conditions, proteins in the lysates of the cells treated with CoCl2 and/or oligomycin A were subjected to 8.5 or 12.5% SDS-PAGE. After electrophoresis, the proteins were transferred to PVDF membranes (Immobilon-FL, Merck-Millipore, Cork, Ireland) . The membrane was blocked with 5% skim milk (Snow Brand, Japan) and incubated with the primary antibodies overnight at 4 °C followed by incubation with the suitable secondary antibody. The primary antibodies used were anti-Oct-4A (9B7) mouse monoclonal IgG (1: 1000, #4286, Cell Signaling Technology, MA), anti-HIF-1α mouse monoclonal IgG1 clone # 241,809 (1: 1000, R&D Systems, MN), anti-CD31 rabbit polyclonal antibody (1:500, ab28364, Abcam, UK), anti-mouse β-tubulin rabbit polyclonal antibody (1: 1000, #2146 s, Cell Signaling Technology), anti β-actin mouse monoclonal antibody (1:2000, 010–27,841, Wako) and anti-GFP goat polyclonal antibody labeled with HRP (1: 2000, ab6663, Abcam, UK). The secondary antibodies used were anti-rabbit IgG goat IgG linked with HRP (1:10,000, #7074 s, Cell Signaling Technology)and anti-mouse IgG goat IgG linked with HRP (1: 10,000, #7076 s, Cell Signaling Technology). HRP reaction was performed with Ez West Lumi plus (ATTO, Japan), and the developed fluorescence intensity was detected by Light Capture II (ATTO, Japan) or Lumino Graph I (ATTO, Japan).
5
Western Blot Protein Analysis
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For Western blotting, cells were lysed in PBS with 2% Triton X-100 containing Pierce protease and phosphatase inhibitor tablets (Thermo Fisher Scientific). The protein concentration was determined with a bicinchoninic acid assay kit (Thermo Fisher Scientific), and equal amounts were resolved on NuPAGE 4–12% precast gels (Invitrogen). Blotting was performed on polyvinylidene fluoride membranes (Immobilon-FL, EMD Millipore) followed by detection using the Odyssey infrared scanning system (LI-COR Biosciences).
6
Optimized Western Blot Procedures
7
Protein Isolation and Western Blot Analysis
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Pi, Si, and MP were isolated from acinar cells and suspended in loading buffer containing SDS and dithiothreitol and denatured in boiling water for 5 min. Aliquots containing 30 mg protein were resolved by electrophoresis on 15% polyacrylamide gels. Proteins were then transferred to Immobilon-FL® membranes (EMD Millipore). Strips representing lanes with replicate MP samples were cut and probed with sera from diseased female NOD mice or from each of the immunized rabbits. After washing, the strips were probed with goat anti-mouse IgG or goat anti-rabbit IgG labeled with infrared fluorescent dye 800. Images were captured with an Odyssey® Scanning Infrared Fluorescent Imaging System (Li-Cor, Lincoln, NB).
8
Western Blot Quantification Protocol
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Cells were lysed in SDS sample buffer and separated by SDS-PAGE. Proteins were transferred to Immobilon-FL PVDF membranes, blocked in Blok-FL (Merck Millipore, Billerica, MA, USA) and left overnight at 4°C in Blok-FL with antibodies diluted 1/1,000. Membranes were washed in Tris-buffered saline containing 0.01% (vol./vol.) Tween 20 and incubated with secondary antibodies (1/10,000 dilution). Immunodetection was performed in Odyssey Imaging System (LI-COR, Lincoln, NE, USA) and band intensities quantified by densitometric scanning using the Odyssey V3.0 software (LI-COR).
9
Immunoprecipitation and Western Blot Analysis of Cobl-ASAP1 Complex
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HEK293T cells transiently coexpressing 3xFLAG-Cobl and GFP-ASAP1 constructs for 24 h were lysed in lysis buffer (25 mM Tris, pH 7.4, 5% glycerol, 150 mM NaCl, 50 mM NaF, 0.1 mM Na3VO4, 10 mM β-glycerol phosphate, 8.7 mg/ml paranitrophenylphosphate, 0.3% Triton X-100, and protease inhibitor tablet [Roche, Indianapolis, IN]) and immunoprecipitated with M2 FLAG resin (Sigma-Aldrich) for 2 h. Immunoprecipitates were than washed four times in wash buffer (lysis buffer but with 0.2% Triton X-100 and no protease inhibitor tablet) and eluted from the FLAG resin with 200 μg/ml 3xFLAG peptide for 15 min at room temperature. Eluates were than denatured with Laemmli buffer, resolved by SDS-PAGE, transferred to Immobilon-FL (EMD Millipore, Billerica, MA), blotted with specific antibody, and visualized by enhanced chemiluminescence.
To determine percentage of Cobl knockdown, Western blots were probed with specific antibody and IRDye 680– or 800–conjugated secondary antibodies. The blot was than imaged using an Odyssey infrared imaging system (LI-COR Biosciences). Analysis to determine percentage Cobl knockdown was performed using Image Studio Lite 4.0 (LI-COR Biosciences). Samples were normalized to the E-cadherin loading control, and knockdown was determined as a percent of the control.
To determine percentage of Cobl knockdown, Western blots were probed with specific antibody and IRDye 680– or 800–conjugated secondary antibodies. The blot was than imaged using an Odyssey infrared imaging system (LI-COR Biosciences). Analysis to determine percentage Cobl knockdown was performed using Image Studio Lite 4.0 (LI-COR Biosciences). Samples were normalized to the E-cadherin loading control, and knockdown was determined as a percent of the control.
10
Western Blot Analysis of Apoptosis Regulators
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After washing with PBS, ca. 3 × 106 cells were directly lysed in pre-heated H8-Buffer (20 mM Tris/HCl pH 7.5, 2 mM EGTA, 2 mM EDTA, 1% SDS, supplemented with 50–100 mM DTT) and boiled at 95 °C for 10 min. The lysates were subsequently homogenized and boiled again for 5 min after addition of 4 × Lämmli buffer (supplemented with 100 mM DTT). Proteins were separated on 12.5 or 15% denaturing SDS-PAGE gels and transferred to PVDF membrane (Immobilon-FL, 0.45 μM, Merck Millipore, Zug, Switzerland). After blocking, the membranes were probed overnight with the following primary antibodies: mouse anti-BCL-2 (clone 10C4, BioLegend); rat anti-BIM (clone 3C5) and rat anti-MCL-1 (clone 19C4); kind gifts from D Huang (Parkville, Victoria, Australia), rabbit polyclonal anti-BCL-XL from Santa Cruz (S-18, Dallas, TX, USA); rabbit polyclonal anti-pro-caspase-3 (#9662), anti-cleaved-caspase-3 (#9661) and polyclonal anti-PUMA (#7467) from Cell Signaling (Danvers, MA, USA); mouse anti-tubulin (clone B-5-1-2) from Sigma Aldrich (St. Louis, MO, USA). For all immunoblots with total lysates, infrared dye-conjugated secondary antibodies (LI-COR Biosciences, Bad Homburg, Germany) were used. All immunoblots were analyzed with the Odyssey Fc Dual-Mode Imaging System using the ImageStudio software 3.1.4 (LI-COR).
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