Western blot analyses were performed to detect AIF and caspase-3 expression on muscle tissue (n=8). Muscle samples were placed in loading buffer, boiled for 5 min and centrifuged. Following quantification, the supernatants were loaded on a 10% sodium dodecylsulfate-polyacrylamide gel and subjected to electrophoresis. The fractionated proteins were transferred onto a polyvinylidene fluoride (PVDF) membranes (Milipore, Billerica, MA, USA), and the membranes, after blocking in 10% non-fat dry milk in TPBS buffer for 1 h at room temperature, were incubated with the primary antibodies to AIF (diluted 1:1,000), caspase-3 (diluted 1:1,000) and β-tubulin (diluted 1:1,000) (all from Cell Signaling Technology, Inc.) and then for 2 h with horseradish peroxidase-conjugated secondary antibodies (1:500; Jackson ImmunoResearch Inc.). After intervening washes, the membranes were developed with the ECL western blotting detection system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the resulting chemilumnescence was exposed to film (Agfa HealthCare, Greenville, SC, USA). A tonsil served as a positive control.
Ecl western blotting detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ECL Western blotting detection system is a laboratory equipment used for the detection and analysis of proteins in Western blot experiments. It utilizes a chemiluminescent reaction to generate a light signal that can be captured and quantified, allowing for the visualization and measurement of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Versadoc 5000 mp imaging system, by Bio-Rad (6 mentions)
β actin, by Merck Group (5 mentions)
Laemmli buffer, by Bio-Rad (4 mentions)
Bca assay kit, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
0.2 m polyvinylidene difluoride membrane, by GE Healthcare (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Bca assay, by Beyotime (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Lysis buffer, by iNtRON Biotechnology (1 mentions)
34 protocols using ecl western blotting detection system
1
Western Blot Analysis of Apoptosis Proteins
2
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum proteins or purified standard antigens were separated by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were probed with individual primary antibodies followed by incubation of HRP-conjugated anti-mouse or anti-rabbit secondary antibodies. The signals were then visualized with the ECL Western Blotting Detection System (ThermoFisher Scientific, USA).
3
Western Blot Analysis of Cell Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from the cultured cells and was quantitated using a BCA assay (Beyotime, Haimen, China). Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with anti-E-cadherin (1:500), anti-vimentin (1:500), anti-HBx (1:200), anti-ZEB2 (1:200) and anti-GAPDH (SCBT, 1:2000) at 4 °C overnight, and then they were incubated with the respective secondary antibody at 37 °C for 2 h. Resulting bands were detected by an ECL Western blotting detection system (Thermo Scientific, Rockford, IL, USA).
4
Western Blot Analysis of ZO-1 and β-Actin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with lysis buffer (INTRON Biotechnology, Daejeon, Korea) containing a protease inhibitor cocktail (Sigma-Aldrich). Proteins in the lysates were separated by SDS-PAGE and transferred onto PVDF membranes. The membrane was separated for binding of ZO-1 (about 220 kDa) and β-actin (about 45 kDa) antibodies. After incubating for 1 h with 5% skim milk in 0.1% Tween 20/TBS to block the non-specific binding of primary antibodies, the separated membranes placed in each tray were probed with specific primary antibodies at 4 °C overnight. After three washes with the wash buffer (0.1% Tween 20/TBS) for 15 min each, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Membranes were then washed a further three times using the wash buffer for 15 min each, and bands were detected using the enhanced chemiluminescence (ECL) Western blotting detection system (Thermo Scientific, IL, USA). Chemiluminescence images were captured using an Amersham Imager 600 (GE Healthcare Life Sciences, Pittsburgh, PA, USA).
5
Western Blot Analysis for Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell lysates (10–30 μg) were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membrane. The membrane was incubated with specific primary antibody overnight followed by the horseradish peroxidase (HRP)-conjugated secondary antibody, and visualized by the ECL Western blotting detection system (Thermo Scientific; Rockford, IL). β-actin and vinculin were both purchased from Sigma (St Louis, MO) and were used as the loading control. Antibodies against α-SMA, SMAD3 and Fibronectin were purchased from Dako (Carpinteria, CA), Santa Cruz (Dallas, TX) and Millipore (Billerica, MA).
6
Quantifying Protein Expression via Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentration was determined with a BCA assay kit (Thermo Scientific, Rockford, IL). Equal amounts of protein were boiled in Laemmli buffer (Bio-Rad, Bio-Rad, Richmond, CA, USA) and loaded onto 12% SDS-PAGE gel and transferred to a 0.2 µm polyvinylidene difluoride membrane (GE Healthcare, Boulder, CO). The membranes were incubated with appropriate primary antibodies for overnight at 4 °C and were then incubated with the appropriate secondary antibodies for 1 h. Detection was performed using the ECL Western blotting detection system (Thermo Scientific, Rockford, IL). The immunoblot was analyzed with a Bio-Rad imaging system (Versadoc 5000 MP Imaging System, Bio-Rad, Richmond, CA, USA).
7
Western Blot Analysis of ARPE-19 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ARPE-19 cells were homogenized and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology). Then, protein concentrations were measured with a BCA protein assay kit (Pierce, Rockford, IL, USA). The proteins (30 µg/lane) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to Immobilon P EMD Millipore (Billerica, MA, USA). After blocking with 5% non-fat milk in PBS with Tween-20 buffer at room temperature for 1 h, the blots were incubated for 60 min at room temperature with primary antibody against the following: Bcl-2, Bax, Akt, phosphorylated Akt, GSK-3β, phosphorylated GSK-3β or GAPDH (diluted 1:1,000 in TBST; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, the membranes were incubated with horseradish-peroxidase-conjugated secondary antibody (1:1,000; Santa Cruz Biotechnology) for 1 h at room temperature. Detection was performed using the ECL western blotting detection system (Thermo Fisher Scientific, Inc.). Each band density was quantified using Quantity One software (Bio-Rad, Richmond, CA, USA) and normalized to GAPDH. The experiment was performed in triplicate.
8
Western Blot Protein Quantification and Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentration was determined with a BCA assay kit (23225; Thermo Scientific, Rockford, IL, USA). Equal amounts of protein were boiled in Laemmli buffer (Bio-Rad, Bio-Rad, Richmond, CA, USA) and loaded onto 12% SDS-PAGE gel and transferred to a 0.2 µm polyvinylidene difluoride membrane (GE Healthcare, Boulder, CO, USA). The membranes were incubated with appropriate primary antibodies overnight at 4 °C and were then incubated with the appropriate secondary antibodies for 1 h. Detection was performed using the ECL Western blotting detection system (Thermo Scientific, Rockford, IL, USA). The immunoblot was analyzed with a Bio-Rad imaging system (Versadoc 5000 MP Imaging System, Bio-Rad, Richmond, CA, USA).
9
Grx2 Protein Expression in Eye
10
Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After different treatments, proteins were extracted with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Taiwan) according to the manufacturer’s instructions. Proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (SDS-PAGE gel, Beyotime, China) and transferred to nitrocellulose membranes (Millipore, USA). Five percent skimmed milk was used to block the membranes for 1 h and then the membranes were incubated overnight at 4 °C with the primary antibodies listed in Supplementary Table 2 . Next, the membranes were incubated with respective secondary antibodies (Proteintech, China) for 1 h at room temperature. Peroxidase labeling was visualized via enhanced chemiluminescence labeling using an ECL Western blotting detection system (Thermo Fisher Scientific, Waltham).
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!