Cells were treated with MeCDDA in complete medium for 24 h. Total protein was isolated using RIPA lysis buffer, and the protein concentrations of lysates were determined using the BCA Protein Assay Kit. Equal amounts of each protein were separated on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. Membranes were then immersed in a blocking solution (Tris-buffered saline with Tween-20 (0.01% Tween), and 5% non-fat milk), and the blot was incubated with primary antibodies overnight at 4 °C, followed by a 1-h incubation with horseradish peroxidase–conjugated secondary antibodies. Membranes were developed using the enhanced chemiluminescence detection kit reagent (ECL prime western blotting Substrate, GE Healthcare Life Sciences, Piscataway, NJ, USA) to detect specific proteins. The primary antibodies to Bax, Bcl-2, cytochrome c, p-AKT, AKT, p-mTOR, mTOR, Flag, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). All the densitometric analysis performed in this study was conducted using ImageJ software (National Institute of Health, Bethesda, MD, USA).
Ecl prime western blotting substrate
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
ECL Prime Western Blotting substrate is a chemiluminescent detection reagent used in Western blotting to visualize and quantify proteins. It generates a stable luminescent signal that can be detected using a compatible imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Sc 7294, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Sc 377460, by Santa Cruz Biotechnology (1 mentions)
A5441, by Merck Group (1 mentions)
T per, by Thermo Fisher Scientific (1 mentions)
Gp62 c, by Progen Biotechnik (1 mentions)
Mouse anti ha monoclonal antibody, by Abcam (1 mentions)
Anti mouse and rabbit igg hrp conjugated secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
Anti flag and anti myc antibodies, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
6 protocols using ecl prime western blotting substrate
1
Western Blotting Analysis of Cell Signaling
2
Western Blot Analysis of PD-L1 and NF-κB Signaling
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Samples were prepared for western blot analysis by adding 4× SDS‐PAGE sample buffer and incubating for 5 minutes at 95°C. Proteins (7.5‐9 μg) were separated by 10% SDS‐PAGE and then transferred onto polyvinylidene difluoride membranes (Bio‐Rad, Tokyo, Japan). Proteins were detected by probing with the following primary antibodies: a rabbit anti‐PD‐L1 (E1L3N) mAb (#13684; CST, Japan), a rabbit anti‐NF‐κB p65 (C22B4) mAb (#4764; CST) or a rabbit anti‐phospho‐NF‐κB p65 (Ser536) (93H1) mAb (#3033; CST), followed by incubation with an HRP‐conjugated anti‐rabbit secondary antibody (Santa Cruz Biotechnology, USA). The membranes were developed using ECL Prime Western Blotting substrate (GE Healthcare, Amersham Place, UK) according to the manufacturer's instructions. Signals were detected on a LAS 4000 EPUV Mini imaging detection system (FUJIFILM, Tokyo, Japan).
3
Western Blot Analysis of Protein Expression
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Samples for Western blotting were collected and stored in T-PER (Thermo Fisher Scientific) at −80 °C until use. The samples were homogenized in ice-cold T-PER with Cell Destroyer (Bio Medical Science), and the total protein concentration was determined using BCA protein assays (Thermo Fisher Scientific). Equal amounts of protein (3 to 5 µg of protein sample per well) were loaded on 10% sodium dodecyl sulfate–polyacrylamide electrophoresis gels (SuperSep Ace) and transblotted to polyvinylidene fluoride membranes. The membranes were incubated in blocking buffer (Blocking One, nacalai tesque) for 1 h and then overnight with primary antibodies, including mouse anti-NFATc1 at 1:5,000 dilution (sc-7294, Santa Cruz); rabbit anticalcineurin at 1:5,000 dilution (GTX59619, CST); mouse anti-MyoD at 1:1,000 dilution (sc-377460, Santa Cruz); guinea pig anti-p62/SQSTM1 at 1:5,000 dilution (GP62-C, PROGEN); and mouse antiβ-actin at 1:5,000 dilution (A5441, Sigma-Aldrich). Primary antibodies were detected by incubation with the appropriate secondary antibody for 1 h at room temperature and then with the ECL Prime Western Blotting Substrate (GE Healthcare) for 5 min. Protein expression was measured using WSE-6100LuminoGraph I (ATTO). The densities were measured using ImageJ software (NIH).
4
SDS-PAGE and Western Blot Analysis of HCT116 and DLD1 Cells
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Pellets of HCT116 and DLD1 cells were resuspended and lysed in 2X Laemmli sample buffer at 98°C for 15 min. The lysed cells were then centrifuged at 117,000 g for 10 min at 16°C to remove insoluble material. SDS-PAGE and Western blotting were performed as described [17 (link)]. The protein samples were separated using 4–20% SDS-PAGE or Bolt™ 8% Bis-Tris gels (Invitrogen) and then blotted onto PVDF membrane. The membrane was blocked in 5% nonfat milk for 1 h before incubation with the primary antibody overnight at 4°C. Then, membrane was rinsed and probed for 1 h with the secondary anti-mouse or anti-rabbit antibodies conjugated to HRP with dilution 1:10,000 in 5% nonfat milk for 1 h. Detection of the signal was performed using ChemiDoc Imaging System with ECL Prime Western Blotting substrate (GE Healthcare).
5
TCA-based Whole Cell Extraction and Immunoprecipitation
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Whole cell extracts were prepared using the trichloroacetic acid (TCA) method as previously described (39 (link)). Immunoprecipitation was carried out following our published method (39 (link)). Proteins from whole-cell extracts or immunoprecipitated samples were resolved on a SDS-PAGE gel and transferred onto a PVDF membrane (Millipore) using a semi-dry transfer cell (Bio-Rad). Mouse monoclonal anti-HA antibody was purchased from Abcam, anti-Myc and anti-FLAG antibodies were purchased from Sigma, and the phosphor-specific rabbit polycolonal antibody against Fun30 phosphorylation on serine 28 was ordered from GenScript (Nanjing). Anti-mouse and rabbit IgG HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology. Blots were developed using the ECL Prime Western Blotting substrate (GE Healthcare).
6
Western Blot Analysis of Protein Expression
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Cells were lysed in ice cold cell lysis buffer (Cell Signaling Technology) supplemented with protease and phosphatase inhibitor cocktails (Roche) according to the manufacturer's instructions. Lysates were pelleted for 15 min at 13 000 xg at 4 °C and supernatants kept for protein quantification (BCA Protein Assay Kit, Thermo Scientific). Equal amounts of cellular proteins were resolved on 10% or 12% sodium dodecyl sulfatepolyacrylamide gels (SDS-PAGE) and subsequently transferred to nitrocellulose membranes (Bio-Rad Laboratories).
Membranes were blocked using 5% non-fat dry milk or 5% bovine serum albumin (BSA) (Sigma-Aldrich) for phosphorylated proteins immunoblots in Tris-buffered saline (TBS) (50 mM Tris, 150 mM NaCl, pH = 7.6) containing 0.1% Tween-20 (Sigma-Aldrich) (TBS-T) for 1 h and primary antibodies were then added in blocking solution. The following antibodies were used: mouse anti rhodopsin 1D4 (Abcam) and mouse anti-β-Actin (Sigma-Aldrich). Primary antibody incubations were carried out at 4°C overnight. After washing with TBS-T, the appropriate HRP-conjugated secondary antibody was added (1:5000 in blocking buffer) for 2 h at room temperature. Antibody binding was detected using chemiluminescence ECL Prime Western Blotting Substrate (GE Healthcare).
Membranes were blocked using 5% non-fat dry milk or 5% bovine serum albumin (BSA) (Sigma-Aldrich) for phosphorylated proteins immunoblots in Tris-buffered saline (TBS) (50 mM Tris, 150 mM NaCl, pH = 7.6) containing 0.1% Tween-20 (Sigma-Aldrich) (TBS-T) for 1 h and primary antibodies were then added in blocking solution. The following antibodies were used: mouse anti rhodopsin 1D4 (Abcam) and mouse anti-β-Actin (Sigma-Aldrich). Primary antibody incubations were carried out at 4°C overnight. After washing with TBS-T, the appropriate HRP-conjugated secondary antibody was added (1:5000 in blocking buffer) for 2 h at room temperature. Antibody binding was detected using chemiluminescence ECL Prime Western Blotting Substrate (GE Healthcare).
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