ab36810 staining human colon tissue. Staining is localized to the nucleus.
Left panel: with primary antibody at 4 µg/ml. Right panel: Isotype control.
Sections were stained using an automated system DAKO Autostainer Plus at room temperature. Sections were rehydrated and antigen retrieved with the DAKO 3-in-1 antigen retrieval buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H₂O₂ in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with hematoxylin and coverslipped under DePeX.

Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

All lanes : Anti-ATM (phospho S1981) antibody [10H11.E12] (ab36810) at 10 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Extract from patient with Ataxia-Telangiectasia whole cell lysate
Lane 3 : Irradiated HeLa Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1 & 3 : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Lane 2 : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under non-reducing conditions.

Predicted band size: 370 kDa
Observed band size: 370 kDa
Additional bands at: 100 kDa, 110 kDa, 145 kDa, 200 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes

Overlay histogram showing HeLa cells stained with ab36810 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab36810, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-ATM (phospho S1981) antibody [10H11.E12] (ab36810) at 1/1000 dilution

Lane 1 : Control lane
Lane 2 : Irradiated Human fibroblasts (10 Gy gamma-irradiation)
Lane 3 : Molecular weight marker
Lane 4 : Peroxidated Human fibroblasts (300 µM hydrogen peroxide)
Lane 5 : Peroxidated Human fibroblasts (1 mM hydrogen peroxide)
Lane 6 : Peroxidated Human fibroblasts (10 mM hydrogen peroxide)

Developed using the ECL technique.

Predicted band size: 370 kDa
Observed band size: 370 kDa



Lysate loading concentration: 40µg