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

Immunofluorescent analysis of HeLa cells treated with H2O2, using Phospho-Histone H2A.X (S139) Antibody.