Protein samples were resuspended in 2× SDS-sample buffer, denatured at 95°C for several minutes and separated by SDS-PAGE according to standard procedures. For western blotting, proteins were transferred to nitrocellulose membranes. Membranes were blocked in TBST buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20) supplemented with 10% dry milk powder overnight at 4°C and incubated for 60–90 min at room temperature with respective primary antibodies in blocking solution. After three 10 min washes in TBST buffer, the membranes were incubated with corresponding peroxidase-coupled secondary antibodies. Bound antibodies were detected with chemiluminescent substrate (Western Lightning® Plus, Perkin Elmer, Waltham, MA) and visualized with iBright™ CL1000 (Thermo Fisher Scientific, Life Technologies, Darmstadt, Germany). Figs 6 , 7 and Fig. S3 show cropped blots. Full, uncropped pictures of the respective blotting membranes are shown in Fig. S4 (blot transparency). Relative protein expression levels were quantified with ImageJ version 1.53n as was described above for mRNA levels. SUN3 protein levels were normalized to the relative amount of actin in the same probes. Test for statistical significance was performed with two-tailed one-sample t-tests using GraphPad Quickcalcs (https://www.graphpad.com/quickcalcs/oneSampleT1/ ).
Western lightning plus
Manufactured by PerkinElmer
Sourced in United States
The Western Lightning Plus is a chemiluminescent detection reagent for western blotting analysis. It is designed to detect and visualize target proteins on nitrocellulose or PVDF membranes.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Na934v, by GE Healthcare (2 mentions)
Cat 8884, by Cell Signaling Technology (2 mentions)
Ibright cl1000, by Thermo Fisher Scientific (2 mentions)
Non fat dry milk, by Bio-Rad (2 mentions)
Fuji super rx film, by Fujifilm (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Nbp1 55368, by Bio-Techne (2 mentions)
Phgdh, by Cell Signaling Technology (2 mentions)
Lamin a c, by Santa Cruz Biotechnology (2 mentions)
β actin, by Merck Group (2 mentions)
Nupage system, by Thermo Fisher Scientific (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Ab183742, by Abcam (1 mentions)
Sc 58052, by Santa Cruz Biotechnology (1 mentions)
Sc 101058, by Santa Cruz Biotechnology (1 mentions)
39 protocols using western lightning plus
1
Western Blot Analysis of Protein Samples
2
Western Blot Analysis of Protein Expression
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Protein was prepared in NuPAGE Sample Buffer and Reducing Agent (Life Technologies, Carlsbad, CA, USA) using 10 μg (estrogen-related blots), 65 μg (V5 blot, T47D-ELF5-isoform 2-V5) or 25 μg (V5 blots, all other lines) per lane. Samples were separated on precast 15-well 4–12 % Bis-Tris (estrogen-related blots) or 10-well 10 % Bis-Tris (V5 blots) polyacrylamide gels (Life Technologies), transferred to polyvinylidene fluoride membrane, blocked in 5 % skim milk, and incubated overnight at 4 °C in primary antibody. Secondary horseradish peroxidase–conjugated antibody was added 1:2000 in 5 % skim milk (anti-mouse, NA931V, anti-rabbit, NA934V; GE Healthcare Life Sciences, Little Chalfont, UK). Proteins were detected using enhanced chemiluminescence solution (Western Lightning Plus; PerkinElmer, Waltham, MA, USA) and x-ray film (Fujifilm, Tokyo, Japan). Primary antibodies used were anti-V5 (sc-58052, 1:500–1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-transducin-like enhancer of split 1 (anti-TLE1) (ab183742, 1:1000; Abcam, Cambridge, UK), anti-ERα (sc-8005, 1:1000; Santa Cruz Biotechnology), anti-Forkhead box A1 (anti-FOXA1) (sc-101058, 1:1000, Santa Cruz Biotechnology), and anti-β-actin (AC-15, 1:20,000; Sigma-Aldrich, St. Louis, MO, USA).
3
Western Blot Analysis of LC3 Protein
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Pellets from SIN-1 treated cells (2x106 cells) were dissolved in Laemmli sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 50 mmol/L DTT, 2% w/v SDS, 20% v/v glycerol, 0.2% w/v bromophenol blue), sonicated (8 s, 30% amplitude) and boiled (5 min, 95 °C). Proteins were separated by SDS-PAGE (15%-Tris-Tricine-Gel; BioRad) and blotted onto a PVDF membrane (0.2 µm GE Healthcare, Buckinghamshire, England; Trans-Blot® Cell, BioRad). Membranes were blocked overnight (4 °C, 5% dry milk in TPBS), incubated with primary mouse monoclonal anti-LC3 antibody (3 h, 1:250 in blocking buffer, RT) and washed (TPBS). Peroxidase-conjugated goat anti-mouse antibody (1 h, 1:10,000 in blocking buffer, RT) was added. Membranes were washed and developed using Western Lightning Plus (Perkin Elmer, Waltham, USA) and Lucent Blue X-ray film (Advansta, Menlo Park, CA, USA). Images were quantified using ImageJ.
4
Western Blot Analysis Protocol
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After samples were run on 4–20% gels, they were transferred to nitrocellulose membranes. Membranes were blocked in 5% (w/v) nonfat dry milk (Bio-Rad, Hercules, CA, USA) in TRIS-buffered saline (TBS) with 0.1% (v/v) Tween-20 (TBST) and then incubated with primary antibodies in 1% (w/v) nonfat dry milk. After washing with TBST, membranes were incubated with horseradish peroxidase-linked secondary antibodies. Membranes were washed with TBST and then TBS before incubation with Western Lightning Plus (PerkinElmer, Waltham, MA, USA) enhanced chemiluminescence reagent, followed by CCD imaging (iBright CL1000, ThermoFisher Scientific). The primary antibody against GAPDH (Cell Signaling Technology Cat 8884, RRID:AB11129865) was conjugated to horseradish peroxidase, so there was not a need for incubation with secondary antibodies.
5
Recombinant Protein Expression Validation
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Recombinant protein expression and quantification were confirmed using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), with bovine serum albumin (BSA) protein standards (KPL, Gaithersburg, MD, USA) for reference. Protein identity was verified by Western blotting using an anti-His-tag monoclonal antibody. The procedure involved loading the protein onto a 10% SDS-PAGE gel, followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Merck, Darmstadt, Germany). The membranes were then incubated with a 6× His tag antibody (GeneTex, Hsinchu, Taiwan) at a 1:5000 dilution as the primary antibody and a rabbit anti-mouse antibody conjugated to horseradish peroxidase (HRP) (GeneTex, Hsinchu City, Taiwan) at a 1:2000 dilution as the secondary antibody. Color development was achieved using Western Lightning PLUS (PerkinElmer, Waltham, MA, USA).
6
Protein Quantification and Western Blot Analysis
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Cell lysate was mixed with 5-fold diluted Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA) to measure the absorbance value. The cell lysate (20 μg) was used for the Western blot analysis with 3-5 mL of primary antibodies: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (MAB374, EMD Millipore, Billerica, MA, USA), anti-mitochondria antibody (ab92824), and SOD2 (ab13533). Signals were detected by adding Western Lightning Plus (PerkinElmer, Waltham, MA, USA). A digital imaging system (Bio Pioneer Tech Co., New Taipei City, Taiwan) was used to detect the signals, which were further analyzed using ImageJ® software.
7
Western Blot Analysis of Myotubes
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Myotubes were lysed in radioimmunoprecipitation assay (RIPA) buffer, homogenised using an Ultra-Turrax (IKA; Staufen, Germany) and denatured in Laemmli buffer for 10 min at 65 °C. Proteins were resolved by SDS–PAGE, electro-transferred and immunoblotted as previously described (Patel et al., 2012 (link)). Specific bands were detected using chemoluminescence (Western Lightning Plus, Perkin Elmer) on Fuji Super RX film (Bedford, UK), scanned and quantified using Quantity One software (BioRad Laboratories, Hemel Hempstead, UK). Loading controls of corresponding total protein immunoreactivity or β-actin were utilised. All treatment groups were represented on each blot on which bands were quantified.
8
Western Blot Analysis of Key Signaling Proteins
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Cells were lysed in buffer (50mM TRIS, 0.5% sodium deoxycholate, 1.0% NP-40, 0.1% SDS, 150mM NaCl, 2mM EDTA) supplemented with protease inhibitors (Roche). Lysates (15-30μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the following antibodies: MYC (sc-40, Santa Cruz), MYCL (#AF4050, R&D), MYCN (#9405, Cell Signaling), Omomyc, p21 (#2947, Cell Signaling), p27 (sc-1641, Santa Cruz), p16 (51-1325GR, BD), PARP1 (#9542, Cell Signaling), TP73 (sc-7957, Santa Cruz), α-Tubulin (CP06, CalBiochemicals). Membranes were then incubated with a peroxidase-conjugated antibody. Enhanced chemiluminescence was performed according to manufacturer's instructions (Western Lightning Plus, Perkin Elmer).
9
Purification and Analysis of HtrA1 Protein
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Ni-NTA columns (Qiagen) were loaded with HtrA1 conditioned medium containing 20 mM imidazole and 0.05% NP-40, washed three times (20 mM imidazole, 0.05% NP-40/PBS) and bound fractions were eluted (200 mM imidazole, 0.05% NP-40/PBS). Eluted fractions were subjected to centrifugal filter devices (Amicon) four times to remove excess imidazole and to concentrate the proteins (2000 g, 30 min, 4°C). Purified protein was reconstituted in PBS.
Samples were resolved on SDS-PAGE (4–20% gradient, Bio-rad) in the presence or absence of reducing agent (355 mM β-mercaptoethanol), transferred onto PVDF membrane (Bio-rad), blocked (5% non-fat dry milk/140 mM NaCl/10 mM Tris pH 8 (TBS-T)) and incubated in primary antibody (1∶1000 in 0.1% sodium azide/3% BSA/TBS-T) overnight (4°C). After washing, membranes were incubated in HRP-conjugated secondary antibodies (1∶5000 in 5% non-fat dry milk/TBS-T). Signal was detected using Western Lightning Plus (Perkin Elmer).
Samples were resolved on SDS-PAGE (4–20% gradient, Bio-rad) in the presence or absence of reducing agent (355 mM β-mercaptoethanol), transferred onto PVDF membrane (Bio-rad), blocked (5% non-fat dry milk/140 mM NaCl/10 mM Tris pH 8 (TBS-T)) and incubated in primary antibody (1∶1000 in 0.1% sodium azide/3% BSA/TBS-T) overnight (4°C). After washing, membranes were incubated in HRP-conjugated secondary antibodies (1∶5000 in 5% non-fat dry milk/TBS-T). Signal was detected using Western Lightning Plus (Perkin Elmer).
10
Protein Extraction and Western Blotting
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Protein from C2C12 cells were extracted using a mammalian lysis buffer (50 mM HEPES pH 7.0, 150 mM NaCl, 10% glycerol, 1% Triton X‐100, 10 mM sodium pyrophosphate, 100 mM NaF, 1 mM EGTA, 1.5 mM MgCl2). Immunoblotting analyses were performed using soluble fraction of whole protein lysates run on 7.5–12% acrylamide gels. Proteins were blotted on to nitrocellulose membranes, blocked with either 10% skim milk or 5% BSA in TRIS‐buffered saline, 0.1% Tween (TBS‐T) for 30–60 min, and probed with antibodies against iNOS (1:5,000 – 1:3,000), and pAMPK (1:5,000 – 1:1,000), AMPK (1:5000 – 1:1,000), pACC (1:5000–1:1,000), ACC (1:1,000), pS6K (1:2,000), S6K (1:5,000), pS6 (1:5,000), S6 (1:1,000), and α‐tubulin (1:5,000). After washing with TBS‐T, blots were subsequently probed with the appropriate horseradish peroxidase‐conjugated secondary antibodies (1:5,000) and exposed to ECL reagent (Western Lightning Plus, Perkin Elmer). Signal was determined by exposure to photosensitive films. Quantifications of Western blot band signal density were performed using ImageJ software (Schneider et al, 2012 ).
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