Cells were lysed 30 min–1 h on ice in RIPA buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.6, 1% NP-40, 0.5% sodium deoxycholate, 5 mM EDTA, supplemented with 1x protease inhibitor cocktail (Sigma), and 1 mM PMSF) or in NP40 lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet-P40, supplemented with 1 mM PMSF, and 1x protease inhibitor cocktail (Sigma)) for co-immunoprecipitation analysis. Cell debris and nuclei were removed by centrifugation for 10 min at 20,000g, 4 °C. Protein levels were determined by DC Protein Assay (Bio-Rad). After SDS-PAGE and blotting on nitrocellulose membranes (GE Healthcare), we used Lumi-Light Western Blotting Substrate (Roche) or SuperSignal West Femto (Thermo Fisher Scientific) and either ECL Hyperfilm (Amersham) or Image Lab (Biorad) for chemiluminescent detection of bands. Signal intensities were quantified using the quantification function of Image Lab (Bio-Rad).
Lumi light western blotting substrate
Manufactured by Roche
Sourced in Switzerland, Germany, United States, Japan
Lumi-Light Western Blotting Substrate is a chemiluminescent substrate used for detecting and quantifying proteins in Western blotting analysis. It is designed to produce a luminescent signal upon reaction with the enzyme-labeled detection antibody.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (5 mentions)
Supersignal west femto, by Thermo Fisher Scientific (4 mentions)
Typhoon 9400 variable mode imager, by GE Healthcare (4 mentions)
β actin, by Merck Group (4 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Protran pure 0.2micron nitrocellulose, by PerkinElmer (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase hrp conjugated goat secondary antibody against rabbit or mouse igg, by Abcam (3 mentions)
Bca kit, by Merck Group (3 mentions)
Las 3000 luminescent image analyzer, by Fujifilm (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Nitrocellulose membrane, by GE Healthcare (3 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Ecl plus reagent, by PerkinElmer (2 mentions)
Las 4000 mini luminescence image analyzer, by Fujifilm (2 mentions)
Protein sample buffer, by Beyotime (2 mentions)
Blocking buffer, by Beyotime (2 mentions)
Versadoc imaging system, by Bio-Rad (2 mentions)
107 protocols using lumi light western blotting substrate
1
Cell Lysis and Immunoblotting Protocol
2
Western Blot Analysis of Atxn3 in Mouse Retina
3
Western Blot Imaging and Analysis
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Lysates and immunoprecipitated proteins were separated on SDS/PAGE gels and transferred on to nitrocellulose membranes (Bio-Rad) for 2 h at 80 V in transfer buffer supplemented with 10% methanol. Membranes were blocked with Odyssey Blocking Buffer (LiCor) and probed with the indicated antibodies. Membranes were washed with PBST and membrane-bound proteins were detected by infrared (LiCor) imaging. Images were recorded as TIFF files for quantification. Band intensities were measured using FIJI [52 (link)]. For presentation, original TIFF files were inverted and contrast enhanced using Adobe Photoshop. Merged colour images were pseudocoloured using Adobe Photoshop.
For crosslinking experiments, samples were run on a SDS/6% PAGE and transferred on to a PVDF membrane for 3 h at 90 V in transfer buffer supplemented with 20% methanol. The membrane was blocked in 5% milk in PBST and EGFP-EVH2 protein was detected on film using the mouse αGFP antibody and Lumi-Light western blotting substrate (Roche).
For crosslinking experiments, samples were run on a SDS/6% PAGE and transferred on to a PVDF membrane for 3 h at 90 V in transfer buffer supplemented with 20% methanol. The membrane was blocked in 5% milk in PBST and EGFP-EVH2 protein was detected on film using the mouse αGFP antibody and Lumi-Light western blotting substrate (Roche).
4
Protein Expression Analysis via Western Blot
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A total of 50 embryos were collected in 10 μL of lysis buffer (RIPA buffer supplemented with a cocktail of protease inhibitors) and stored at –80 °C. Samples were then mixed with protein sample buffer (Beyotime, China) and heated at 95 °C for 5 min. Proteins were separated using a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) system (Tanon, China) at 100 V for 120–150 min. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes with an electrophoretic transfer apparatus (Tanon, China) at 65 V for 120 min. Thereafter, membranes were blocked in blocking buffer (Beyotime, China) for 2 h and then incubated with primary antibodies at 4 °C overnight. After washing, the membranes were incubated with secondary antibodies at RT for 1.5–2 h. Signals were detected with a Lumi-Light Western Blotting Substrate (Roche, Germany) and images were acquired using a VersaDoc Imaging System (Bio-Rad, USA). The signal intensity of bands was measured as integrated intensity with Image J and normalized to background intensity. Details on primary and secondary antibodies used in this study are provided in Supplementary Tables S2, S3.
5
Western Blot Analysis of Signaling Proteins
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Denatured protein lysates were resolved by 4–12 % NuPAGE gels (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes (Schleicher & Schuell, Dachen, Germany). The membranes were incubated with anti-5-LO (BD, Franklin Lakes, NJ); anti-p-Akt (Ser473), anti-pan-Akt (Cell signaling, Danvers, MA); or monoclonal anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature or overnight at 4 °C. Membranes were then washed (4 times) with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL) for 1 h. Immunoreactive proteins were visualized by developing them with Lumi-light western blotting substrate (Roche Diagnostics GmbH, Mannheim, Germany), followed by exposure in a LAS-3000 (Fuji Film Co., Tokyo, Japan) according to the manufacturer’s instructions. This was followed by quantitation of specific bands with the Multi Gauge software (Fuji Film Co., Tokyo, Japan).
6
Western Blot Analysis of WT1 Protein
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Total protein collection was performed using reagent TRIzol according to the manufacturer's instructions. Protein samples (50 μg) were electrophoresed on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Immunodetection of WT1 protein isoform was performed using a WT1C19 polyclonal antibody (COOH-terminal binding, Santa Cruz Biotechnology); the β-actin monoclonal antibody was obtained from Sigma Chemical (St Louis, MO), and anti-mouse and anti-rabbit antibodies conjugated with horseradish peroxidase were purchased from Bio-Rad. Protein bands were visualized by enhanced chemiluminescence using Roche Lumi-Light Western blotting substrate. Subsequently, the bands obtained were analyzed by densitometry using the ImageJ program (https://imagej.nih.gov/ij/download.html ). The density of each band was normalized with its respective value of β-actin.
7
Western Blot Analysis of Apoptosis Markers
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Cells were lysed with cell lysis buffer (CST, MA, USA; Cat #9803) in the presence of protease inhibitors. 40 μg of total protein were electrophoresed on 12% SDS-PAGE and electrophoretically transferred onto a PVDF membrane, blocked with 5% skim milk at room temperature (RT) for one hour. Membranes were later probed with different primary antibodies overnight at 4 °C. The membranes were washed for 5 min three times in TBS with 0.1% Tween-20 and then incubated with horseradish peroxidase-conjugated mouse (Cat #1706516, Biorad, CA, USA) or rabbit (Cat #1706515, Biorad, CA, USA) secondary antibodies at RT for one hour. The membranes were washed three times for 5 min in TBS with 0.1% Tween-20, and then visualized with the Lumi-Light Western Blotting Substrate (Roche, Basel, Switzerland; Cat #12015200001) on the 5200 chemiluminescence imager (Tanon, Shanghai, China). The following primary antibodies were purchased: mouse anti-PTGES (Santa Cruz, CA, USA; Cat #sc-166,309), rabbit anti-Cleaved Caspase-3 (CST, MA, USA; Cat #9661), mouse anti-Cleaved PARP-1 (Santa Cruz, CA, USA; Cat #sc-56,196), mouse anti-GAPDH (ZSGB-BIO, Beijing, China; Cat #TA-08).
8
Western Blot Analysis of TCV P38 Protein
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To detect TCV P38 protein, the anti-TCV P38 antibody and the horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG secondary antibody (Invitrogen) was used at 1:10,000 dilution. To detect Flag-tagged P38 and derivative proteins, the monoclonal anti-Flag M2-HRP antibody (Sigma-Aldrich) was used at 1:8000 dilution. The chemiluminescent signals induced by Lumi-Light Western Blotting Substrate (Roche) were detected using X-Ray film (FUJIFILM) and CURIX 60 processor (AGFA).
9
Western Blot Analysis of Atxn3 in Mouse Retina
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Retina lysates from Atxn3+/+ and Atxn3−/− mice were obtained by homogenization and sonication in RIPA lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.5% NP40, with protease inhibitors (Complete, Roche Diagnostics, Indianapolis, IN)). Proteins from Atxn3+/+ and Atxn3−/− mouse RPE were extracted following a previously described protocol (Wei et al., 2016 (link)). Proteins (30 μg) were resolved in 10%–12.5% SDS-PAGE gels, transferred onto PVDF membranes and blocked with 5%–10% non-fat milk in PBS-T for 1 h, followed by an overnight incubation at 4°C with primary antibodies and a peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:2000) for 1 h. Bands were visualized by treatment either with the ECL-plus reagent (Western Lightning®, PerkinElmer, Waltham, MA) and exposure to autoradiography films or with Lumi-Light Western Blotting Substrate (Roche Diagnostics, Indianapolis, IN) and chemiluminescence detection in a LAS-4000 mini Luminescence Image Analyzer (Fujifilm, Tokyo, Japan). Fiji software (Schindelin et al., 2012 (link)) was used to quantify protein band density and α-tubulin or GAPDH were used for normalization. Figure 4 shows representative western blots (n = 4). Uncropped blots are shown in Figure S5D .
10
Western Blot Analysis of Protein Interactors
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Cells were lysed in cold FLAG-IP buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM EDTA, 1% Triton X-100) supplemented with protease and phosphatase inhibitors and centrifuged at 15,000 × g for 10 min at 4 °C. Supernatant was collected as whole cell extract (WCE). Equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and incubated for 4 h in 5% milk blocking buffer. The membranes were incubated with SMYD5, USP11, α-Tubulin, GAPDH, Flag, V5 or Tat antibodies overnight, followed by incubation with a secondary α-rabbit or α-mouse antibody conjugated to HRP at a dilution of 1:10000 for 1 h. Blotted membranes were developed by using Lumi-Light Western Blotting Substrate (Roche) or SuperSignal™ West Femto (Thermo Fisher Scientific) and imaged with Amersham Hyperfilm ECL or ChemiDoc Imaging Systems (Bio-Rad). All western blots were performed at least three times.
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