Immunoblot analysis was performed according to an established protocol (8 (link)). The expression of FLIP were recognized by incubation overnight at 4°C with rabbit monoclonal antibody to mouse FLIP (Cell Signaling Technology), followed by anti-rabbit secondary antibodies conjugated with horseradish peroxidase (GE healthcare). After stripping, the same membrane were blotted with antibody to GAPDH (Sigma-Aldrich) for loading adjustment. The specific proteins were detected employing the Enhanced Chemiluminescent Detection Reagent (Amersham Pharmacia).
Enhanced chemiluminescent detection reagent
Manufactured by Cytiva
Sourced in United States
The Enhanced chemiluminescent detection reagent is a laboratory tool designed to facilitate the detection and visualization of proteins in Western blot analysis. It utilizes a chemiluminescent reaction to generate a luminescent signal, allowing for the sensitive and quantitative detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase, by GE Healthcare (1 mentions)
Antibody to gapdh, by Merck Group (1 mentions)
Anti aml1, by Cell Signaling Technology (1 mentions)
Anti α actin, by Merck Group (1 mentions)
Anti acetyl histone h3, by Merck Group (1 mentions)
Anti rabbit hrp, by Agilent Technologies (1 mentions)
Anti mouse horseradish peroxidase hrp, by Agilent Technologies (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Mini trans blot electrophoretic transfer cell, by Bio-Rad (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
4 protocols using enhanced chemiluminescent detection reagent
1
Immunoblot Analysis of FLIP Protein
2
Western Blot Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were prepared using cell lysis buffer (150 mmol/L NaCl, 10 mmol/L Tris-HCl [pH 7.4], 5 mmol/L EDTA, 1% Triton X-100) supplemented with PMSF and the cOmplete protease inhibitor cocktail (EDTA-free; Roche). Lysates were subjected to SDS-PAGE and transferred to PVDF membrane (Millipore). Membranes were blocked in skimmed milk or BSA and probed overnight with the following antibodies: anti-AML1 (Cell Signaling Technology; cat. #4336); anti-acetyl-histone H3 (Millipore; cat. #06-599); anti-histone H3 (Abcam; cat. #1791); and anti-α-actin (Sigma; cat. #AC-74). Filters were then washed, and probed with either anti-mouse horseradish peroxidase (HRP) or anti-rabbit HRP (both DakoCytomation) secondary antibodies. Signals were visualized using enhanced chemiluminescent detection reagent (Amersham).
3
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins or cell lysates were separated on 10% (w/v) SDS-PAGE acrylamide gels and stained using Coomassie Brilliant Blue, or transferred onto a Whatman Protran nitrocellulose membrane at 100 V for 1 h using a mini Trans-Blot® Electrophoretic Transfer Cell (BioRad). Membranes were blocked for 1 h with 5% (w/v) skim milk (Diploma, Australia) in TBS-Tween [5 mM Tris-HCl, 15 mM NaCl, pH 7.4, 0.05% (v/v) Tween20]. The membranes were then washed twice (15 min wash followed by a 5 min wash) in TBS-Tween, incubated with a primary antibody in TBS-Tween for 1 h, washed again as before, incubated with a secondary antibody conjugated to horseradish peroxidase and washed as before. All incubations and washes were performed with shaking at room temperature. Membranes were developed with an enhanced chemiluminescent detection reagent (Amersham Life Science) in a FPM-100A film developer (FujiFilm).
4
Immunoblot Analysis of HAND1 and HAND2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblot analysis was performed as previously described
[14 (link)]. Cell lysates were separated with SDS-PAGE in 10% polyacrylamide gels and transferred onto nitrocellulose membranes. After blocking of nonspecific binding sites for 2 hours with 5% nonfat milk in PBS with 0.1% Tween-20, we incubated the membranes with 1:1,000 anti-HAND1 polyclonal antibody or 1:1,000 anti-HAND2 monoclonal antibody (Abcam, Cambridge, MA, USA); anti-β-actin antibody (Sigma-Aldrich, St Louis, MO, USA; dilution 1:100,000) was used as protein loading control. The proteins were detected by using Enhanced Chemiluminescent detection reagent (Amersham, Piscataway, NJ, USA), according to the manufacturer’s instructions.
[14 (link)]. Cell lysates were separated with SDS-PAGE in 10% polyacrylamide gels and transferred onto nitrocellulose membranes. After blocking of nonspecific binding sites for 2 hours with 5% nonfat milk in PBS with 0.1% Tween-20, we incubated the membranes with 1:1,000 anti-HAND1 polyclonal antibody or 1:1,000 anti-HAND2 monoclonal antibody (Abcam, Cambridge, MA, USA); anti-β-actin antibody (Sigma-Aldrich, St Louis, MO, USA; dilution 1:100,000) was used as protein loading control. The proteins were detected by using Enhanced Chemiluminescent detection reagent (Amersham, Piscataway, NJ, USA), according to the manufacturer’s instructions.
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!