Cells or tissue was lysed with EDTA-free lysis buffer (Roche Applied Science, Cat.04719946001) with 1 × protease/phosphatase inhibitor cocktail (Cell Signaling, #5872). Protein concentrations were measured with BCA protein assay kit (Thermo Scientific, 23227), separated with NuPAGE bis-tris Precast gels (Life technologies), and transferred to PVDF membrane with an iblot Western blotting system (Life Technologies) according to the manufacturer's instructions. Membranes were blocked using 5% blotting grade blocker (Bio-Rad #170-6404). Antibodies used included polyclonal anti-ADAR1 (1:1,000; Abcam), anti-CHOP (1:1,000; Abcam ab170379), monoclonal anti-GAPDH (1:3,000; Chemicon), anti-IRE-1alpha (1:1,000; Abcam ab37073), followed by corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2,500) and ECL detection (Sigma).
Ecl detection
Manufactured by Merck Group
Sourced in United States
ECL detection is a laboratory technique used to measure the presence and quantity of specific proteins or molecules in a sample. It utilizes a chemiluminescent reaction to generate light, which is then detected and quantified using specialized equipment. The core function of ECL detection is to provide a sensitive and accurate method for protein analysis and quantification in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Nupage bis tris precast gel, by Thermo Fisher Scientific (2 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Immobilon, by Merck Group (2 mentions)
Pepstatin, by Merck Group (2 mentions)
E cadherin, by BD (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Merck Group (1 mentions)
Edta free lysis buffer, by Roche (1 mentions)
Iblot western blotting system, by Thermo Fisher Scientific (1 mentions)
Anti adar1, by Abcam (1 mentions)
Iblot2 western blotting system, by Thermo Fisher Scientific (1 mentions)
Blotting grade blocker, by Bio-Rad (1 mentions)
P ampkα, by Abcam (1 mentions)
Ripa buffer, by BestBio (1 mentions)
20 protocols using ecl detection
1
Western Blot Analysis of ADAR1, CHOP, and IRE-1α
2
Western Blot Analysis of Cellular and Tissue Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or cardiac tissue were lysed with lysis buffer (50 mm Tris-HCL pH 7.4, 100 mm NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with 1x protease/phosphatase inhibitor cocktail (Cell Signaling, #5872). Protein concentrations were measured with BCA protein assay kit (Thermo Scientific, 23227), 5 μg cell lysate or 20 μg tissue lysate was loaded on Nupage bis-tris Precast gels (Life Technologies), and transferred to PVDF membrane with iblot 2 Western blotting system (Life Technologies), according to manufacturer instructions. Membranes were first blocked with 5% blotting grade blocker (Bio-Rad #170-6404). After washing, primary antibodies were diluted in 5% TBST and applied to the membrane overnight at 4C. After washing, appropriate horseradish peroxidase HRP-conjugated secondary antibodies were used for enhanced chemiluminescent (ECL) detection (Sigma). All the antibodies used in these studies and their dilution are listed in Supplementary Table 2 .
3
Western Blot Analysis of Kidney Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The frozen kidney tissue was homogenized in RIPA buffer (BestBio, Shanghai, China), and the protein content was determined by the BCA method. Equal amounts of protein were separated on 10% SDS-PAGE. The proteins were then transferred onto the PVDF membrane and were blocked using 5% skimmed milk. The membranes were incubated with primary antibodies all night at 4°C and then with secondary antibodies at room temperature. The enhanced chemiluminescence (ECL) detection (Millipore, MA, USA) was utilized to make the membranes visualized. At last, the destination band was scanned and quantified using the infrared fluorescence imaging system (Odyssey, USA).
The primary antibodies used in this study are as follows: AMPKα (1 : 800, Cell Signaling Technology, USA, 5831), p-AMPKα (1 : 1000, 2535), Nrf2 (1 : 1000, Abcam, USA, 89443), Nox4 (1 : 1000, 133303), TGF-β1 (1 : 600, 215715), HO-1 (1 : 1000, Santa Cruz, USA, 390991), NQO1 (1 : 1000, 376023), and NFκB (1 : 600, Servicebio, China, 11997).
The primary antibodies used in this study are as follows: AMPKα (1 : 800, Cell Signaling Technology, USA, 5831), p-AMPKα (1 : 1000, 2535), Nrf2 (1 : 1000, Abcam, USA, 89443), Nox4 (1 : 1000, 133303), TGF-β1 (1 : 600, 215715), HO-1 (1 : 1000, Santa Cruz, USA, 390991), NQO1 (1 : 1000, 376023), and NFκB (1 : 600, Servicebio, China, 11997).
4
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and clinical tissues were lysed for 30–45 mins on ice in lysis buffer containing freshly added protease inhibitor cocktail (Roche Diagnostics, USA). After BCA quantification (Pierce, USA), the protein was added to 5× loading buffer and boiled at 95°C for 10 min. Equal amounts of protein were separated by SDS-PAGE and blotted onto activated polyvinylidene difluoride membranes (Millipore, USA). After blocking, the membranes were incubated with primary antibodies overnight at 4°C. The antibodies used were as follows: BMX (1:2000, 610793, BD), phospho-Etk (Tyr40) (1:500, #3211, Cell Signaling Technology), AKT1 (1:500, sc-5298, Santa Cruz Biotechnology), p-AKT (1:1000, #4060, Cell Signaling Technology), mTOR (1:1000, A2445, ABclonal), p-mTOR (1:1000, AP0094, ABclonal), STAT3 (1:1000, #9132, Cell Signaling Technology), phospho-STAT3 (Tyr705) (1:1000, #4113, Cell Signaling Technology), β-actin and GAPDH (1:1000, Santa Cruz Biotechnology). Blots were incubated with secondary antibodies coupled to horseradish peroxidase (Thermo Fisher Scientific, USA) for 1 h and visualized using ECL detection (Millipore) on X-ray film. Relative quantitation was analyzed with Quantity One software.
5
Immunoblot Analysis of Mitochondrial OXPHOS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Preparation of tissue lysate was carried out as described previously [93 (link)]. From this, 10 μg protein was loaded into 4–20% gradient gels (Mini-PROTEAN® TGX™ Precast Protein Gels, 15-well, 15 μl #4561096, BioRAD) alongside a protein ladder (Precision plus protein dual color standards #1610374, BioRAD). The gel was transferred to nitrocellulose membrane, and this was stained in Ponceau S. The membrane was blocked in 5% Skimmed milk-TBS-T for 1 h prior to primary antibody incubation (Total OXPHOS antibody cocktail, ab110412, Abcam, RRID:AB_2847807; 1:500 in 1% Milk TBS-T) overnight at 4 °C. The membrane was incubated with secondary antibody (Rabbit anti-Mouse IgG HRP, #61-6520, Invitrogen; RRID:AB_2533933 1:5000 in TBS-T)) for 1 h at room temperature, before ECL detection (Milipore) and imaging using iBright 1500 (ThermoFisher Scientific). Band density was quantified using Image J software [92 (link)]. The original immunoblot image is presented in Additional File 1 : Figure S8H.
6
Western Blot Analysis of ZO-1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell cultures were rinsed with PBS (pH = 7.4) and lysed in RIPA buffer (50 mmol/L Tris–HCl, 30 mmol/L NaCl, 1% NP40, 10 mmol/L NaF, 2 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, complete protease inhibitor cocktail (Roche Applied Science), 1 mmol/L EDTA, 0.05% sodium dodecyl sulphate, 5 mmol/L sodium-deoxycholate, pH = 7.4). Cell lysates were gently mixed at 4°C for 20 min, and then spun at 13.500 rpm for 20 min to collect the supernatant. The concentration of the isolated proteins was determined using BCA Protein Assay Reagent (Thermo Scientific). About 10 μg of protein was separated by SDS–PAGE and electrophoretically transferred to PVDF membranes (Immobilon, Millipore). After 2 h blocking with 5% milk and 1% Tween in PBS, the membranes were incubated with primary antibodies recognizing ZO-1 (rabbit polyclonal, ThermoFisher Scientific, 1/250) or GAPDH (mouse monoclonal IgG, EMD Millipore, 1/30000). Thereafter, the appropriate secondary horseradish peroxidase-conjugated antibodies (Jackson ImmunoResearch; 1/5000) were used, followed by ECL detection (Millipore) using ImageQuant LAS4000 software. Band intensities were quantified using NIH Image software (NIH AutoExtractor 1.51; National Institutes of Health).
7
Western Blot Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SW620inv cells were used for ICW analysis as described [10 (link)]. Whole cells were analyzed in RIPA buffer (150mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 50mM Tris-Cl, pH = 8.0) supplemented with a protease inhibitor cocktail consisting of 1μg/mL aprotinin, 1μg/mL leupeptin, 3μg/mL Pepstatin, 1mM NaVO3, 1mM NaF, 0.5 μM DTT (Sigma Chemical, St. Louis, MO). Protein was separated by 10-15% polyacrylamide gels, depending on the size of the protein of interest. Antibodies used in this study are as follows: E-cadherin (BD Transduction Laboratories, Franklin Lakes, NJ), SNAIL and SLUG (Cell Signaling), β-actin and GAPDH (Sigma Chemical), HNF4α and both anti-mouse and anti-rabbit secondary antibodies are from Santa Cruz. Western blots have been done by regular ECL detection (Millipore) or Odyssey IR imaging system (LI-COR Biosciences) [10 (link)].
8
Cx40 Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell cultures were rinsed with PBS, pH = 7.4, and lysed in RIPA buffer as previously described (Pfenniger et al., 2012 (link)). After protein concentration quantification with a Micro BCA protein assay kit (Thermo Scientific), 5 μg (HUVECs) or 10 μg (bEnd.3) of protein was separated by SDS-PAGE and transferred to PVDF-membrane (Immobilon, Millipore). After 2 h blocking with 5% milk and 1% Tween in PBS, the membrane was exposed to anti-Cx40 (Alpha-Diagnostics, 1/500) or anti-β-actin (Sigma, 1/10000) primary antibodies in blocking solution. Revelation was performed by incubating the membrane for 1 h at RT with secondary horseradish peroxidase-conjugated antibodies (Jackson ImmunoResearch; 1/5000) and followed by ECL detection (Millipore) using ImageQuant LAS 4000 software. Band intensities were thereafter quantified using ImageJ software. Cx40 results were normalized to β-actin.
9
Analysis of Autoantibody Levels in Thyroid Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment with MMI and/or Se for 24 h, the thyroid cells were harvested and lysed in a RIPA and PMSF mixture (RIPA:PMSF = 99:1). The total protein concentrations were determined by using a bicinchoninic acid kit (Beyotime, Jiangsu, China). Then equal amounts of proteins (50 µg/lane) were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with different primary antibodies, rabbit anti-TRAb (1:400, Boster, Wuhan, China), rabbit anti-TPOAb (1:400, Boster), or rabbit anti-TGAb (1:400, Boster), overnight at 4 °C. The immunoblots were then incubated with antirabbit IgG-horseradish peroxidase (1:4000, Boster) as a secondary antibody followed by ECL detection (Millipore, Billerica, MA, USA).
10
Western Blot Analysis of DNA Damage Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA buffer containing 100 mM Tris, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 1 mM EDTA, 0.5% NP-40, 0.1% SDS, 0.2% deoxycholate, 25 mM β-glycerophosphate, 10 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO3, 10 mM NaF, 1× protease inhibitor cocktail 2, and 1× phosphatase inhibitor. After sonication, cell lysates containing 20 μg of protein were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking with 5% heated non-fat milk, membranes were incubated with primary antibody overnight at 4 °C. Antibodies used are: R1(Santa Cruz), R2(Santa Cruz), p53R2(Santa Cruz), CHK2 (Millipore), pCHK2 (Thr68; Cell signaling), thymidylate synthase (TS, ZYMED), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; GeneTex), thymidine kinase 1(TK1)35 (link), and β-Tubulin (Sigma). Membranes were then treated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) for 1 hr, followed by ECL detection according to the manufacturer’s instructions (Millipore).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!