DCs cocultured with or without Th9 cells were harvested and lysed with lysis buffer containing protease inhibitor. Proteins were separated on a 12% polyacrylamide gel, and blotted onto nitrocellulose membranes. Mouse monoclonal Ab against Akt was obtained from Cell Signaling, and Ab against phosphorylated (p)-Ark was obtained from Santa Cruz. Rabbit polyclonal Abs against p38 MAPK, STAT5, cleaved (c)-Caspase-3, Bcl-xL, and Bax were obtained from Cell Signaling. Rabbit monoclonal Abs against p-p38, ERK, p-ERK, and p-STAT5 were obtained from Cell Signaling. Rabbit anti-β-actin polyclonal Ab was obtained from Sigma-Aldrich. After blocking with 5% nonfat milk protein/PBS, membranes were incubated with primary Abs and visualized with horseradish peroxidase-conjugated secondary Abs (Bio-rad) and an ECL substrate detection system (Amersham Pharmacia).
Horseradish peroxidase conjugated secondary abs
Manufactured by Bio-Rad
Sourced in United States
Horseradish peroxidase-conjugated secondary antibodies are detection reagents used in various immunoassay techniques. They consist of secondary antibodies that are chemically linked to the enzyme horseradish peroxidase. This enzyme can catalyze a color-producing reaction, allowing for the visualization and detection of target proteins or molecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
P stat5, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence western blotting substrate, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
Anti bcl2 associated x bax, by Santa Cruz Biotechnology (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
2 protocols using horseradish peroxidase conjugated secondary abs
1
Immunoblot Analysis of Dendritic Cell Signaling
2
Western Blot Analysis of Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from tissues and cells using radioimmunoprecipitation assay buffer (catalog No. 8900; Thermo Fisher Scientific) containing a cocktail of protease and phosphatase inhibitors (GenDEPOT, Baker, TX, USA). The protein lysates (20 μg) were separated using 10%–12% SDS-PAGE and transferred (100 volts, 90 min) onto polyvinylidene difluoride membranes (GE Healthcare, Solingen, Germany). Membranes were blocked using 5% skim milk with Tris-buffered saline containing 0.1% Tween 20 for 1 h and then probed with the primary Abs, anti-Miro-1 (catalog No. NBP1-89011; Novus Biologicals, Centennial, CO, USA), anti-Bcl-2 (catalog No. sc-7382; Santa Cruz Biotechnology), and anti-Bcl-2-associated X (Bax) (catalog No. sc-7480; Santa Cruz Biotechnology), overnight at 4°C. The membranes were incubated with horseradish peroxidase-conjugated secondary Abs (anti-rabbit, catalog No. 1706515 or anti-mouse, catalog No. 1706516; Bio-Rad, Hercules, CA, USA) for 1 h at 20–25°C. The signal was developed using an enhanced chemiluminescence western blotting substrate (Bio-Rad). Quantification was performed using the ImageJ software and normalized against β-actin.
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!