Samples:

Nuclear extract (50 µg) from human HEK293, human melanoma (G361), mouse wildtype embryonic fibroblasts (+/+) or mouse H2AX knockout embryonic fibroblasts (-/-). Antibody: ab11174 used at 0.1 mcg/ml. Detection: Chemiluminescence with 30 second exposure.

UT = untreated
Ub = ubiquitylated
NCS = neocarzinostatin - 200 ng/ml, 30 min

 

Samples: Nuclear extract (50 µg) from human HEK293, human melanoma (G361), mouse wildtype embryonic fibroblasts (+/+) or mouse H2AX knockout embryonic fibroblasts (-/-). Antibody: ab11174 used at 0.1 mcg/ml. Detection: Chemiluminescence with 30 second exposure.

UT = untreated
Ub = ubiquitylated
NCS = neocarzinostatin - 200 ng/ml, 30 min

Immunocytochemistry/Immunofluorescence analysis of neocarzinostatin treated asynchronous HeLa cells (left) and untreated asynchronous HeLa cells (right) labelling H2A.X (phospho S139 with ab11174 at 1/5000 (0.2µg/ml). A DyLight® 594-conjugated anti-rabbit IgG (1/100) was used as the secondary antibody.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labelling gamma H2A.X (phospho S139) with ab11174 at 1/5000 (0.2µg/ml). Dectection: DAB.

ab11174 at 0.5ug staining gamma H2A.X in human Jurkat cell line by flow cytometery. Cells were treated with 5ug/ml etopside for 3 hours, fixed in 1.5% paraformaldehyde, and permeabilized in 90% methanol. A FITC-conjugated rabbit polyclonal was used as secondary (in 150ul reaction). The black line indicates cells treated with etopside and anti KLH antibody, the red line repersents untreated cells and anti gamma H2A.X and blue line indicates etopside treated anti gamma H2A.X respectively.

All lanes : Anti-gamma H2A.X (phospho S139) antibody (ab11174) at 1/5000 dilution

Lane 1 : HeLa nuclear lysate - untreated
Lane 2 : HeLa nuclear lysate - IR treated

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : HRP-conjugated donkey anti-rabbit IgG polyclonal

Developed using the ECL technique.

Performed under reducing conditions.

Observed band size: 17 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

See Abreview

Detection of gamma-H2AX by Immunofluorescence. Samples: Wildtype (H2AX +/+) or H2AX knockout (H2AX -/-) mouse embryonic fibroblasts. Antibody: Affinity purified ab11174 used at 2ug/ml. Detection: Rhodamine red labelled goat anti-rabbit IgG.

ab11174 at 1/1000 staining human HeLa cells by ICC/IF. These cells express a gene that causes a DNA damage response, leading to H2AX phosphorylation. The cells were paraformaldehyde fixed and blocked with BSA prior to incubation with the antibody for 45 minutes. An Alexa-Fluor ®  488 conjugated goat anti-rabbit was used as the secondary.

See Abreview