Cells were plated in a six-well plate to 70% to 80% confluency. After allowing cells to settle overnight, media was replaced with serum-free media for 24 hours. Cells were treated with DMSO or increasing concentrations of test compound for 24 hours. Cells were harvested in RIPA lysis buffer combined with protease and phosphatase inhibitors. Protein yield was assessed using Pierce BCA Protein Assay Kit (Thermofisher, catalog No. 23225) and quantified using a spectrophotometer plate reader (TECAN) at 562 nm. A total of 20-μg protein extracts were loaded per well in 4% to 15% Mini-PROTEAN TGX Precast Protein gels (Bio-Rad, catalog No. 4561084). After electrophoresis, proteins were transferred onto a 0.2 μmol/L nitrocellulose membrane. The membrane was blocked with LICOR Odyssey Blocking Buffer in PBS. After blocking, the membrane was incubated with anti–phospho-histone H2AX (Ser 139) rabbit antibody (Cell Signaling Technology, catalog No. 2577; RRID:AB_2118010) and H2AX rabbit antibody (Abcam, catalog No. ab11175; RRID:AB_297814) at 4°C overnight and incubated with donkey anti-rabbit IRDye800CW secondary antibody (LI-COR, catalog No. 926–32213; RRID:AB_621848) for 1 hour at room temperature. The membrane was washed with 1X TBS with 1% tween-20 (TBS-T) before being scanned with an Odyssey scanner (LI-COR).
Anti phospho histone h2ax ser 139 rabbit antibody
Manufactured by Cell Signaling Technology
The Anti–phospho-histone H2AX (Ser 139) rabbit antibody is a laboratory reagent used to detect the phosphorylation of histone H2AX at serine 139. Histone H2AX phosphorylation is a widely used marker for DNA double-strand breaks.
Automatically generated - may contain errors
2 protocols using anti phospho histone h2ax ser 139 rabbit antibody
1
Quantification of Phospho-H2AX in Cell Lysates
2
Breast Tumor γH2AX Expression in Diabetes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Accumulation of nuclear γH2AX protein was examined in 105 formalin-fixed and paraffin-embedded breast tumor tissues obtained from the Department of Pathology at the University of Maryland Clinical Center. Twenty-nine tumors (17 ER+ [59%], 11 ER– [38%], 1 ER unknown) were obtained from patients with diabetes at time of disease diagnosis and 76 (45 ER+ [59%], 30 ER– [39%], 1 ER unknown) from patients who were nondiabetic at disease diagnosis. We used the anti–phospho-histone H2A.X (Ser139) rabbit antibody from Cell Signaling (catalog 9718) at a 1:800 dilution to visualize γH2AX protein in the tumor sections. Nuclear γH2AX in the tumor epithelium was scored as negative, low, moderate, or high using a standard scoring system as previously described by us and others (72 (link), 73 (link)). Scoring of the IHC was performed by a pathologist blinded to the diabetes status of the patients.
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!