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Anti-Phospho-Histone H2A.X (S139) H2AFX Monoclonal Antibody

Immunofluorescent analysis of HeLa cells treated with H2O2, using Phospho-Histone H2A.X (S139) Antibody.
Figure 1. Western blot analysis of HistoneH2A.X using anti-HistoneH2A.X antibody (MP00241).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human Jurkat whole cell lysates,
Lane 2: human U20S whole cell lysates,
Lane 3: human PC-3 whole cell lysates,
Lane 4: human 293T whole cell lysates,
Lane 5: rat PC-12 whole cell lysates,
Lane 6: rat C6 whole cell lysates,
Lane 7: mouse NIH/3T3 whole cell lysates,
Lane 8: mouse Neuro-2a whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-HistoneH2A.X antigen affinity purified monoclonal antibody (Catalog # MP00241) at 1:5000 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:1000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for HistoneH2A.X at approximately 15 kDa. The expected band size for HistoneH2A.X is at 15 kDa.
Figure 2. IHC analysis of HistoneH2A.X using anti-HistoneH2A.X antibody (MP00241).
HistoneH2A.X was detected in a paraffin-embedded section of human colorectal adenocarcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-HistoneH2A.X Antibody (MP00241) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.
Figure 3. IHC analysis of HistoneH2A.X using anti-HistoneH2A.X antibody (MP00241).
HistoneH2A.X was detected in a paraffin-embedded section of human liver cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-HistoneH2A.X Antibody (MP00241) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.
Figure 4. IHC analysis of HistoneH2A.X using anti-HistoneH2A.X antibody (MP00241).
HistoneH2A.X was detected in a paraffin-embedded section of human placenta tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-HistoneH2A.X Antibody (MP00241) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.
Figure 5. IHC analysis of HistoneH2A.X using anti-HistoneH2A.X antibody (MP00241).
HistoneH2A.X was detected in a paraffin-embedded section of mouse liver tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-HistoneH2A.X Antibody (MP00241) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

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Product Information
Application Applications Legend: E=ELISA WB=Western Blotting IHC=Immunohistochemistry IHC-P=Immunohistochemistry (Paraffin) IP=Immunoprecipitation IF=Immunofluorescence IC=Immunochemistry ICC=Immunocytochemistry FC=Flow Cytometry DB=Dot Blot	WB, IHC, IF, ICC, IP
Primary Accession	P16104
Host	Rabbit
Isotype	Rabbit IgG
Reactivity	Rat, Human, Mouse
Clonality	Monoclonal
Format	Liquid
Description	Anti-Phospho-Histone H2A.X (S139) H2AFX Monoclonal Antibody . Tested in WB, IHC, ICC/IF, IP applications. This antibody reacts with Human, Mouse, Rat.

Additional Information
Gene ID	3014
Other Names	Histone H2AX, H2a/x, Histone H2A.X, H2AX (HGNC:4739)
Calculated MW	15145 Da
Application Details	WB 1:5000-1:10000 IHC 1:50-1:200 ICC/IF 1:50-1:200 IP 1:30
Source	Eukaryota
Contents	Rabbit IgG in phosphate buffered saline, pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol, 0.4-0.5mg/ml BSA.
Clone Names	Clone: AbH41
Immunogen	A synthesized peptide derived from human Phospho-Histone H2A.X (S139)
Purification	Affinity-chromatography
Storage	Store at -20°C for one year. For short term storage and frequent use, store at 4°C for up to one month. Avoid repeated freeze-thaw cycles.

For Research Use Only. Not For Use In Diagnostic Procedures.

Protein Information
Name	H2AX (HGNC:4739)
Function	Variant histone H2A which replaces conventional H2A in a subset of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post- translational modifications of histones, also called histone code, and nucleosome remodeling. Required for checkpoint-mediated arrest of cell cycle progression in response to low doses of ionizing radiation and for efficient repair of DNA double strand breaks (DSBs) specifically when modified by C-terminal phosphorylation.
Cellular Location	Nucleus. Chromosome

Research Areas

Application Protocols

Provided below are standard protocols that you may find useful for product applications.

Western Blot Protocol

Blocking Peptides Protocol

Dot Blot Protocol

Immunohistochemistry Protocol

Immunofluorescence Protocol

Immunoprecipitation Protocol

Flow Cytomety Protocol

Cell Culture Protocol