MCF10A protein was extracted using M-PER containing an EDTA-free Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Massachusetts, USA), while proteins from all other cell lines were extracted using RIPA lysis buffer. Protein transfer was performed on a nitrocellulose membrane (Bio-Rad Laboratories, California, USA). Primary antibodies include: UBE2S (11878; Cell Signaling Technology, Massachusetts, USA; 1:1,000), UBE2C (14234; Cell Signaling Technology; 1:1,000), Numb (sc-136554; Santa Cruz Biotechnology, Texas, USA; 1:200), and ACTIN (3700; Cell Signaling Technology; 1:1,000). Protein was visualized with the ECL Western Blotting Detection System (PerkinElmer, USA), and a densitometry value was determined in terms of pixel intensity by ImageJ.
Ecl western blotting detection system
Manufactured by PerkinElmer
Sourced in United States
The ECL Western Blotting Detection System is a laboratory equipment used for the detection and visualization of proteins in Western blot analysis. It utilizes a chemiluminescent substrate to generate a light signal that can be captured and quantified, allowing for the sensitive and accurate detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ube2s, by Cell Signaling Technology (1 mentions)
Ube2c, by Cell Signaling Technology (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Human il 1β, by Thermo Fisher Scientific (1 mentions)
Anti phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (1 mentions)
Sp600125, by Enzo Life Sciences (1 mentions)
Anti phospho foxo1 ser256, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle s medium dmem f12, by Thermo Fisher Scientific (1 mentions)
Recombinant human tnf α protein, by R&D Systems (1 mentions)
Anti tnfr2, by Santa Cruz Biotechnology (1 mentions)
Anti phospho egfr tyr1173, by Cell Signaling Technology (1 mentions)
Ag1478, by Enzo Life Sciences (1 mentions)
Anti phospho p65 ser536, by Cell Signaling Technology (1 mentions)
Anti phospho jnk1 2 thr183 tyr185, by Cell Signaling Technology (1 mentions)
Helenalin, by Cayman Chemical (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
As1842856, by Merck Group (1 mentions)
Halt protease inhibitor cocktail edta free 100x, by Thermo Fisher Scientific (1 mentions)
5 protocols using ecl western blotting detection system
1
Protein Expression Analysis of MCF10A
2
Protein Expression Analysis of p15, p16, and p21
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LLC cells were collected after LMPs treatment for 24 hours. Extraction of soluble proteins and fractionation by SDS-PAGE was performed as described previously (5 (link)). The anti-p15, anti-p16 and anti-p21 antibodies (LifeSpan BioSciences, Inc. 1:500) were used to reveal the protein levels of p15INK4b, p16INK4a and p21 Cip1, respectively. β-Actin was used as a loading control. Proteins were visualized using the ECL western blotting detection system (PerkinElmer, Inc.). Densitometry values were measured in terms of pixel intensity by Fluorchem software.
3
Protein Extraction and Western Blot
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Cells were cultured to 95% confluency, treated or untreated with IL-1β at different times, as indicated in the descriptions of individual experiments and washed with 1X PBS, and pelleted at 5000× g at 4 °C for 4 min. Cell pellets were lysed with RIPA buffer (1X PBS/1% Nonidet p-40/0.5% sodium deoxycholate/0.1% sodium dodecyl sulfate (SDS)). Cellular debris was removed by centrifugation at 15,000× g for 10 min. The amount of protein in the supernatant solution was determined, and samples were heat-treated in 2X SDS sample loading buffer at 100 °C for 5 min. Equal amounts of samples were fractioned by SDS/PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% non-fat skim milk powder in PBS for an hour and then probed with primary antibodies, which were visualized with horseradish peroxidase-coupled secondary antibodies by using the Enhanced Chemiluminescence (ECL) Western Blotting Detection System (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA). Human IL-1β (Pepro-Tech., East Windsor, NJ, USA) was used at 10 ng/mL.
4
Signaling Pathway Analysis Protocol
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Dulbecco’s modified Eagle’s medium (DMEM)/F-12 and fetal bovine serum were purchased from Thermo Fisher Scientific (Grand Island, NY, USA). BioTraceTM NT membrane was purchased from Pall Life Sciences (Ann Arbor, MI, USA). The enhanced chemiluminescence (ECL) Western blotting detection system was purchased from Perkin Elmer (Waltham, MA, USA). Anti-phospho-EGFR (Tyr1173, Cat#4407), anti-phospho-p38 MAPK (Thr180/Tyr182, Cat#9211), anti-phospho-JNK1/2 (Thr183/Tyr185, Cat#9255), anti-phospho-FoxO1 (Ser256, Cat#9461), and anti-phospho-p65 (Ser536, Cat#3033) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GAPDH (Cat#MCA-1D4) was purchased from EnCor Biotechnology (Gainesville, FL, USA). AG1478, SP600125, and Tanshinone IIA were purchased from Enzo Life Science (Farmingdale, NY, USA). AS1842856 was obtained from EMD Millipore (Billerica, MA, USA). Helenalin was purchased from Cayman Chemical (Ann Arbor, MI, USA). Recombinant human TNF-α protein was purchased from R&D Systems (Minneapolis, MN, USA). TRIzol reagent, enzymes, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
5
Western Blot Analysis of Protein Targets
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Total proteins were extracted from MCF10A using M-PER containing Halt™ Protease Inhibitor Cocktail, EDTA-Free (100X) (Thermo Fisher Scientific, Inc.). Western blot was performed as previously described [22] (link). All antibodies used in this study were purchased from Cell Signaling Technology, Inc., including anti-PGRMC1 (#13,856, l:1000), anti-EGFR (#2085, l:1000), anti-mechanistic target of rapamycin (mTOR) (#2983, l:1000), anti-phosphatase and tensin homolog (PTEN) (#9188, l:1000), and anti-β-actin (#3700, l:1000). β-actin was used as a loading control. Proteins were visualized using an ECL Western blotting detection system (PerkinElmer, Inc.). Densitometry values were measured in terms of pixel intensity by ImageJ.
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