All lanes : Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78) at 1/500 dilution

Lane 1 : 30ug HeLa
Lane 2 : 30ug HeLa nuclear extract
Lane 3 : 30ug SK-N-SH

Secondary
All lanes : Mouse IgG antibody (HRP) at 1/5000 dilution

Predicted band size: 350 kDa

5% SDS-PAGE.

Running conditions: 80V, 15min; 140V, 40 minutes.

Transfer condition: Semi-dry, 18 V, 60 min (NC membrane).

Blocking condition: 5% non-fat milk in TBST, RT, 60 minutes.

Primary antibody incubation: 4°C, overnight.

Washing condition: 5 ml TBST, 4 x 5 minutes. 

Exposure: enhanced ECL

ab78 staining ATM in Human Testis sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Samples were incubated with primary antibody (5ug/ml) and a Biotin-conjugated rabbit anti-mouse IgG was used as the secondary antibody.

Overlay histogram showing HeLa cells stained with ab78 (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 (ab78, 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.

ab78 (2µg/ml) staining ATM in human colonic mucosa, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining of mucosal epithelium and lymphocytes.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Detection of human ATM protein using anti-ATM 2C1 monoclonal antibody (ab78) by western blot.

ab78 staining ATM in Human Kidney sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Samples were incubated with primary antibody (5ug/ml) and a Biotin-conjugated rabbit anti-mouse IgG was used as the secondary antibody.

Detection of human ATM protein using anti-ATM 2C1 monoclonal antibody (ab78) by immunoprecipitation.