Protein lysates were prepared from cell pellets following direct isolation or cell sorting using Pierce radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific; 89900) and standard methods. Lysates were run on 10% Mini PROTEAN TGX precast gels (Bio-Rad Laboratories) and transferred onto 0.2-µm nitrocellulose membranes (Bio-Rad Laboratories; 162-0146). Membranes were blocked in 5% milk and immunoblotted with primary antibody to GIMAP5 (MAC421; Wong et al., 2010 (link); courtesy of Dr. Butcher) overnight at 4°C or with GAPDH (Invitrogen; AM4300) for 1 h at room temperature. Secondary antibodies used were ECL anti-rat IgG HRP-linked whole antibody (GE Healthcare; NA935) and ECL peroxidase-labeled anti-mouse antibody (GE Healthcare; NA931), respectively. Immunostaining was detected using Amersham ECL and ECL Prime Western Blotting detection reagents (GE Healthcare).
Pierce radioimmunoprecipitation assay lysis buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce radioimmunoprecipitation assay (RIPA) lysis buffer is a buffer solution used to extract and solubilize proteins from cells or tissues for further analysis. It contains a combination of detergents, salts, and buffers that aid in the disruption of cell membranes and the release of intracellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Thermo Fisher Scientific (1 mentions)
Ecl peroxidase labeled anti mouse antibody, by GE Healthcare (1 mentions)
0.2 m nitrocellulose membrane, by Bio-Rad (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Amersham ecl, by GE Healthcare (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Thermo Fisher Scientific (1 mentions)
2 protocols using pierce radioimmunoprecipitation assay lysis buffer
1
Protein Lysate Preparation and Western Blot Analysis
2
Western Blot Analysis of Akt and ERK
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were collected after UDCA treatment. Treated cells were lysed with Pierce radioimmunoprecipitation assay lysis buffer (Thermo Scientific, Waltham, MA, USA) containing protease inhibitor. Lysates were then processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were quantified using the Bradford assay (Thermo Scientific), resolved on a 10% Bis-Tris gel, and transferred to a nitrocellulose membrane. The membrane was then blocked for 1 h at room temperature with 5% bovine serum albumin and washed with 1X phosphate-buffered saline-Tween-20 buffer, before being incubated with anti-Akt and -ERK primary antibodies (Cell Signaling Technology, Danvers, MA, USA) for 24 h at 4°C. Immunoreactive bands were detected using anti-immunoglobulin G secondary antibodies and chemiluminescence reagents (Thermo Scientific). Immunoblots were visualized using X-ray film.
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!