Full-length human Polη or Polλ ORFs were cloned into the pCMV7-3xFlag-zeo vector (Sigma-Aldrich) and stably expressed into XPV HFs or GM637 HFs, respectively. For Rev7 foci formation, Rev7 antibody (BD Biosciences) was used for immunofluorescence in GM637 HFs. Cells were suspended and treated with siRNA and cultured on a coverslip in six-well plate with 50% confluence. After 48 h, cells were treated with UVC (30 J/m2). For UV irradiation, cells were washed with PBS buffer and irradiated with UVC light in the presence of PBS buffer. After irradiation, fresh growth medium was added to cells and the cells were incubated for 6 h. After washing with PBS buffer, cells were fixed with 4% paraformaldehyde for 30 min. Fixed cells were permeabilized with 0.2% Triton x-100 in PBS buffer. Primary FLAG antibody (Sigma-Aldrich) or Rev7 antibody (BD Biosciences) were added to cells in PBST (0.1% Tween 20 in PBS) containing 3% BSA. Nuclear staining was performed with DAPI (Molecular Probe) in PBS buffer for 20 min. The fluorescent images were visualized and captured by fluorescence microscopy (Nikon fluorescence microscope).
Rev7 antibody
Manufactured by BD
The Rev7 antibody is a laboratory reagent used in research applications. It is a protein that specifically binds to and identifies a target molecule, enabling its detection and study. The core function of the Rev7 antibody is to serve as a tool for researchers to investigate the target molecule and its biological role. No further details on intended use or interpretation are provided.
Automatically generated - may contain errors
2 protocols using rev7 antibody
1
Evaluating DNA Damage Response Mechanisms
2
Visualization of UV-induced Polη, Polκ, and Rev7 foci in MEFs
Check if the same lab product or an alternative is used in the 5 most similar protocols
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To examine UV-induced Polη and Polκ focus formation in MEFs, Rev1+/+ and Rev1−/− MEFs were immortalized by lentiviral expression of SV40-T antigen (GeneCopoeia). GFP-Polη or GFP-Polκ was transiently transfected into transformed MEFs. After 16 h of incubation, cells were suspended and cultured on a coverslip in a six-well plate with 50% confluence. After 48 h, cells were treated with 20 J/m2 of UVC. After 6 h of incubation, cells were fixed with 4% paraformaldehyde for 30 min. Nuclear staining was performed with DAPI (Molecular Probe) in PBS buffer for 20 min. To examine Rev7 foci in MEFs, primary Rev1+/+ and Rev1−/− MEFs were cultured on a coverslip and incubated for 20 h. Cells were treated with 20 J/m2 of UVC, and, after 6 h incubation, cells were fixed with 4% paraformaldehyde for 30 min. Fixed cells were permeablized with 0.2% Triton X-100 in PBS buffer. Cells were immunostained with Rev7 antibody (BD Bioscience) and then incubated with Alexa fluor 488 secondary antibody (Molecular Probe). Nuclear DNA was stained with DAPI (Molecular Probe) for 20 min. The fluorescent images were visualized and captured by fluorescence microscope (Nikon fluorescence microscope).
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