Nylon strips with embedded phosphatidylinositol (PtdIns) lipids and other phospholipids (100 pmol per spot; Echelon Biosciences) were pre-incubated in blocking buffer [3% bovine serum albumin (BSA; w/v; fatty acid free) in standard phosphate-buffered saline (PBS) pH 7.4 with 0.1% Tween-20 (PBST)] for 2 h at room temperature (rt) on a rocking platform. rSpTrf-E1 or recombinant fragments (~20 nM) were incubated with a lipid-embedded strip in fresh blocking buffer for 2 h at rt with rocking. Unbound proteins were removed with three washes of PBST. Strips were incubated for 2 h at rt with primary antibodies composed of three polyclonal rabbit anti-SpTrf antibodies [anti-SpTrf-66, -68, and -71; 1:3,500 dilution (14 (link), 15 (link))] in blocking buffer, washed, and post-incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (GαRIg-HRP; 1:7,000 dilution in blocking buffer; Thermo Scientific Pierce) for 1 h at rt with rocking. Antibody–protein complexes on washed strips were visualized by incubation with enhanced chemiluminescence Western blotting substrate (Thermo Scientific Pierce) and exposed to autoradiography film (MidSci). Experiments were performed at least three times to confirm binding between rSpTrf-E1 or the recombinant fragments and the phospholipids. Negative controls omitted either rSpTrf-E1, the recombinant fragments, or the primary antibodies.
Enhanced chemiluminescence western blotting substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Enhanced chemiluminescence Western blotting substrate is a laboratory reagent used in the detection and quantification of proteins in Western blotting experiments. It generates a luminescent signal in the presence of the target protein, which can be captured and analyzed using specialized imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
β actin, by Merck Group (3 mentions)
Amersham imager 680 system, by GE Healthcare (3 mentions)
Goat anti mouse igg, by Bio-Rad (3 mentions)
Goat anti rabbit igg, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
To 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Merck Group (2 mentions)
4 to 15 sds polyacrylamide gels, by Bio-Rad (2 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (2 mentions)
Non fat dried milk, by Bio-Rad (2 mentions)
Horseradish peroxidase conjugated secondary antibodies, by Elabscience (2 mentions)
Ripa lysis buffer, by Avantor (2 mentions)
Gradient sds page gel, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Tween 20, by Thermo Fisher Scientific (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
26 protocols using enhanced chemiluminescence western blotting substrate
1
Lipid Binding Assay for Recombinant Proteins
2
Western Blot Analysis of SSC Lysates
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The lysates of SSC were prepared in radioimmunoprecipitation assay lysis buffer (Biomed, Shanghai) supplemented with protease inhibitor cocktail tablets (Roche) and 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 16,000g for 10 min at 4°C, and the supernatants were used for subsequent analyses. After electrophoresis on 10% SDS–polyacrylamide gel electrophoresis gels, proteins were transferred to polyvinylidene difluoride membranes and blocked in 5% skimmed milk. The membranes were incubated with primary antibody (table S7) at 4°C overnight and horseradish peroxidase–labeled secondary antibody (table S7) for 1 hour at 37°C. Proteins were visualized using Enhanced Chemiluminescence Western Blotting Substrate (Thermo Scientific Pierce).
3
Western Blot Analysis of Viral Proteins
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T75 flasks containing confluent MRC-5 cells were infected, and cells were harvested by trypsinization on day five post-infection, lysed in 400 μL Laemmli buffer (Bio-Rad, Hercules, CA, USA), and stored at −20 °C. Aliquots were adjusted to 5% β-mercaptoethanol and heated to 95 °C for five min. prior to separation on 10% Mini-PROTEAN TGX polyacrylamide gels (Bio-Rad) using Tris/glycine/SDS running buffer (Bio-Rad). Proteins were then transferred to a 0.4 μm nitrocellulose membrane (Bio-Rad) using Tris/glycine transfer buffer (Bio-Rad) with 20% methanol. The membrane was blocked for one hour in PBS with 0.02% Tween-20 (Fisher) and 5% powdered milk, then incubated at 4 °C overnight with mouse monoclonal antibodies M2 to FLAG (1:500), MAB810 to CMV IE1 and IE2 proteins (1:500, Fisher Scientific, Hampton, NH, USA), or AC-74 to β-actin (1:2000, Millipore Sigma). After washing with PBS 0.02% Tween-20, the membrane was incubated with a horseradish peroxidase-conjugated goat α-mouse IgG antibody (1:5000, Thermo-Fisher) in blocking buffer for one hour at room temperature, washed with PBS 0.02% Tween-20, and incubated for five min. with enhanced chemiluminescence western blotting substrate (Thermo-Fisher). Chemiluminescence was detected using a ChemiDoc imaging system (Bio-Rad).
4
Quantifying eIF2α Protein Expression via Western Blot
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Protein concentrations from cells collected in RIPA (radioimmunoprecipitation assay) buffer were quantified by bicinchoninic acid assay (Pierce, 23225). Protein (25 µg) was loaded onto a 4–15% MP TGX precast cassette (Bio-Rad, 4561083), and proteins were separated at 100 V for 1 h 25 min. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and were blocked with 5% bovine serum albumin (BSA) in 1× tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1–2 h at room temperature. The eIF2α antibody (1:500; EMD Millipore 07–760-I) was incubated with the PVDF membrane overnight at 4°C in 5% BSA in TBS-T. On the following day, a secondary antibody was applied (donkey anti-rabbit–HRP; Thermo Fisher Scientific; A16023; 1:2,000). Blots were visualized with enhanced chemiluminescence Western blotting substrate (Thermo Fisher Scientific, 32106).
5
Immunoblotting of Protein Expression
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Human embryonic kidney 293 cells were lysed using a Protein Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. The lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were incubated with blocking solution (5% skim milk in Tris-buffered saline with 0.05% Tween-20) for 1 h and then incubated with a blocking solution containing a primary antibody (Ng (Abcam, Cambridge, UK) 1:1000; β-actin (Sigma-Aldrich, St. Louis, MO, USA) 1:5000) overnight at 4 °C. The membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence western blotting substrate (Thermo Scientific).
6
Western Blot Analysis of Cortical Neuron Lysates
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Lysates were made from cultured cortical neurons at 6 DIV in RIPA buffer containing protease and phosphatase inhibitor cocktails as previously described (Brittain et al., 2009 (link)). Samples were boiled in Laemmli sample buffer and separated by electrophoresis on SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes and blocked at room temperature for 1 h and incubated in primary antibodies CRMP2 (Cat# C2993, Sigma, St. Louis, MO), CRMP2 pThr509/Thr514 (Cat# PB-043, Kinasource, Dundee, Scotland, UK), CRMP2 pSer522 (Cat# CP2191, ECM Biosciences, Versailles, KY), GSK3β pSer9 (Cat# 5558, Cell Signaling, Danvers, MA), Cdk5 (Cat# 2506, Cell Signaling, Danvers, MA), p35 (Cat# 2680, Cell Signaling, Danvers, MA), GSK3β (Cat# 9832, Cell Signaling, Danvers, MA), or Tubulin (Cat# G712A, Promega, Madison, WI) overnight at 4°C. Following incubation in horseradish peroxidase conjugated secondary antibodies, blots were probed with enhanced chemiluminescence Western blotting substrate (Thermo Scientific, Waltham, MA) before exposure to photographic film. Films were scanned, digitized, and quantified using Un-Scan-It gel version 6.1 scanning software (Silk Scientific Inc, Orem, UT).
7
Western Blot Protein Analysis Protocol
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A total of 40 μg of protein lysate was run on a 4% to 12% Bis-Tris gel (Thermo Fisher Scientific, Waltham, MA, USA) at 200 volts for 40 min and then transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) in a room set to 4 °C at 40 volts for 14 h [32 (link)]. The membrane was then blocked in a 5% non-fat dry milk solution in tris-buffered saline (TBST, Boston BioProducts, Milford, MA, USA) for 1 h [32 (link)]. The membranes were mixed with primary antibody dilutions in 3% bovine serum albumin in TBST and set to incubate in the 4 °C room for 24 h. The membranes were then removed from the cold room and incubated in horseradish-peroxidase-linked secondary antibody dilutions in 3% bovine serum albumin in TBST for 1 h at room temperature [32 (link)]. The membranes were incubated with the Enhanced Chemiluminescence Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) developing agent and imaged on a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA). Finally, Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific, Waltham, MA, USA) was used to strip membranes for repeat probing if needed for the investigation of additional targets [32 (link)]. Immunoblot band intensity was measured using Image J software version 1.54 (National Institutes of Health, Bethesda, Maryland, USA).
8
Protein Oxidation Detection via Oxyblot
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The MilliporeSigma™ OxyBlot™ Protein Oxidation Detection Kit (Burlington, MA, USA) was used for Oxyblot analysis. A total of 20 μg of protein lysate was run on a 4% to 12% Bis-Tris gel (Thermo Fisher Scientific, Waltham, MA, USA) after mixing the lysate with the kit components in concordance with manufacturer recommendations. The gel was transferred onto a nitrocellulose membrane (Bio-Rad) in the 4 °C room at 40 volts for 14 h. The membrane was incubated with the primary and secondary antibodies provided in the Oxyblot kit at room temperature per the manufacturer’s recommendations. The membrane was incubated with the Enhanced Chemiluminescence Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) developing agent and imaged on a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA) as above. Band intensity was measured using Image J software version 1.54 (National Institutes of Health, Bethesda, Maryland, USA).
9
Immunoblotting Procedure for Protein Analysis
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Cells were washed briefly with 1× PBS and scraped in 1× PBS and collected into tubes. The cells were then resuspended in 1× Laemmli buffer (0.01M Tris (pH 6.8), 1% SDS, 10% glycerol, and 0.1% β-mercapthoethanol). Equal amounts of protein were loaded to 7% SDS-PAGE and transferred to a nitrocellulose membrane for immunoblotting (IB). The membrane was then blocked with 5% not-fat dry milk in 1× PBS + Tween 20 (0.5%). Rabbit anti-TRAP 150 (Bethyl, 1:1000), WU10 (Ref. [8 (link)]; 1:1000), or anti-β-actin (1:3000, Sigma-Aldrich, St. Louis, MO, USA, Cat. No. A5441) were used as primary IB antibodies, followed by secondary horseradish peroxidase (HRP)-conjugated donkey anti-mouse or HRP-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) used at 1:25,000. Enhanced chemiluminescence Western blotting substrate (Thermo Scientific, Waltham, MA, USA) was used to detect HRP. For the cell cycle progression experiments, the following antibodies were used. Mouse anti-tubulin (Sigma, St. Louis, MO, USA 1:2500 for IF or WB), mouse anti-beta-actin (Sigma, 1:3000 for IF or WB), human anti-ACA antibody (Abcam, Cambridge, MA, USA, 1:3000 for IF), mouse anti-Aurora-B (Abcam, Cambridge, MA, USA, 1:100 for IF and WB), and mouse anti- phospho-H3 antibody (Abcam, Cambridge, MA, USA, 1:100 for WB).
10
SARS-CoV-2 Nucleocapsid Protein Expression Analysis
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