Protein-matched aliquots of whole cell lysates were subjected to SDS-PAGE and immunoblot analysis of RAD6 (17), PCNA (Dako, CA), FANCD2, KU86, KU70 (Santa Cruz Biotechnology Inc., TX), POL η (Abcam, MA), γH2AX (BioLegend, CA), RAD18 (Imgenex Corp., CA), RAD51 (Calbiochem, MA), and β-actin (Sigma-Aldrich Chemicals, MO). Since the peptide we used for generating RAD6B antibody is 91% conserved in human RAD6A, the RAD6 proteins detected by our antibody will not distinguish RAD6A and RAD6B proteins, and hence is referred as RAD6 rather than RAD6A or RAD6B [17 (link)]. For PCNA or H2AX ubiquitination analysis, lysates were immunoprecipitated with anti-PCNA, rabbit anti-H2AX (Abcam) or the corresponding normal IgG, and the captured immune complexes subjected to immunoblotting with anti-ubiquitin antibody (Santa Cruz, TX). Stripped membranes were reprobed with PCNA or mouse anti-H2AX antibody (Santa Cruz) to verify PCNA or H2AX pull-down, respectively. H2AX-depleted supernatants were immunoblotted with anti-γH2AX antibody. The relative levels of monoubiquitinated-PCNA, γH2AX and monoubiquitinated-γH2AX were quantified by Image J.
Rabbit anti h2ax
Manufactured by Abcam
Sourced in United States, France
Rabbit anti-H2AX is a primary antibody that recognizes the phosphorylated form of the histone variant H2AX. H2AX is a marker of DNA double-strand breaks and is commonly used to study the cellular response to DNA damage.
Automatically generated - may contain errors
Lab products found in correlation
γh2ax, by BioLegend (1 mentions)
Fancd2, by Santa Cruz Biotechnology (1 mentions)
Anti ubiquitin antibody, by Santa Cruz Biotechnology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Anti pcna, by Abcam (1 mentions)
Mouse anti phospho atm, by Rockland Immunochemicals (1 mentions)
Rabbit anti glyceraldehyde 3 phosphate dehydrogenase anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Alexa fluor 488 labeled donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti tet1, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti γh2ax, by Abcam (1 mentions)
Mouse anti vinculin, by Merck Group (1 mentions)
Nuclear extract kit, by Active Motif (1 mentions)
Rabbit anti gpx4, by Abcam (1 mentions)
Alexafluor 488 donkey anti rabbit antibody, by Abcam (1 mentions)
5 protocols using rabbit anti h2ax
1
Quantitative Analysis of DNA Damage Response Proteins
2
Chromatin Immunoprecipitation Antibody Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibodies used for chromatin immunoprecipitation, immunoblotting, and immunofluorescence staining were rabbit anti-phospho ATM/ATR substrate motif (Cell Signaling Technology, Danvers, USA), mouse anti-phospho ATM (Rockland, Pottstown, USA), mouse anti-HA, mouse anti-γH2AX (Merck Millipore), rabbit anti-RAD51 (Bio Academia,Japan), rabbit anti -RPA2 S4/8 and rabbit anti-INO80 (Bethyl, Montgomery, USA), mouse anti-RPA34 (Lab Vision, Fremont, USA), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (Santa Cruz Biotechnology, USA), rabbit anti-H2AX (Abcam, UK), and mouse anti-β-actin (Sigma-Aldrich) and rabbit anti-ARP8 (Osakabe et al., 2014 (link)).
3
Immunofluorescence Staining of Tet1 and γH2AX
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, washed three times with PBS (5 min each time), permeabilized with 0.25% Triton X-100 for 10 min at room temperature, washed with PBS (three times, 5 min each), and blocked with 5% BSA for 60 min at 37 ℃. The cells were then incubated with the following primary antibodies: rabbit anti-Tet1 (1:500, Abcam) and rabbit anti-H2AX (1:400, Abcam), overnight at 4 ℃. Cells were washed with PBS and probed with Alexa Fluor 488-labeled donkey anti-rabbit IgG (1:1000; Invitrogen) for 60 min at 37 ℃. After three PBS washes (5 min each), the nuclei were stained with DAPI for 10 min. Five microscope fields (× 200) were selected randomly for the evaluation. The mean fluorescence intensity was analysed using the ImageJ software.
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted using nuclear extract kit (Active Motif, La Hulpe, Belgium) according to the manufacturer's guidelines. Western blot was carried out using extracts with adjusted protein concentration (Pierce BCA Protein assay kit, Fisher Scientific, France) with 1:5000 rabbit anti-Hsp27 polyclonal antibody (Stressgen), 1:1000 rabbit anti-γ-H2AX (Abcam), 1:1000 rabbit anti-H2AX (Abcam, Paris, France), and 1:3000 mouse anti-vinculin (Sigma Aldrich, France) used as internal loading control.
5
Evaluation of GPX4 and H2A.X in PANC-1/GEM Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PANC‐1/GEM cells were grown in 12‐well plates to achieve ≈80% confluence. Then, the participants were divided into groups and administered either a placebo or medication; the US group received US therapy for 2 min using a 1 W cm−2 machine. The cells were permeabilized with 0.2% Triton X‐100 on ice for 20 min after being fixed with 4% paraformaldehyde overnight. This was followed by three 10‐min washes in 0.01 m phosphate‐buffered saline. Rabbit anti‐GPX4 (1:200; Abcam) and rabbit anti–H2A.X (1:100; Abcam) were incubated with the cells overnight at 4 °C after being blocked with 2% bovine serum albumin at room temperature for 1 h and rinsed with PBS three times for 10 min each. The cells were treated overnight at room temperature with Alexa Fluor 488 donkey anti‐rabbit antibody (1:1000; Abcam) and washed three times with 0.01 m PBS the following day. The cells were fixed in a medium containing 4′,6‐diamino‐2‐phenylindole (DAPI), and CLSM was used to reconstruct them in three dimensions after three washes in PBS.
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!