Western blot - Rad50 antibody [13B3/2C6] (ab89)

Predicted band size : 146 kDa

This picture was kindly supplied as part of the reviews for ab214 and for ab89 submitted by Anya Polischouk.

The bands represent nuclear (N) and cytoplasmic (C) extract from human lung cancer cells (U1810).

Western blot - Rad50 antibody [13B3/2C6] (ab89)

Anti-Rad50 antibody [13B3/2C6] (ab89) at 1/1000 dilution + 50 ug Human HEK293 total cell lysate

Secondary
HRP conjugated Donkey Anti-Mouse IgG
developed using the ECL technique

Performed under non-reducing conditions.

Predicted band size : 146 kDa
Observed band size : 160 kDa (why is the actual band size different from the predicted?)
Additional bands at : 300 kDa (possible non-specific binding).

Exposure time : 30 seconds

This image is courtesy of an Abreview submitted by Philippe Szankasi on 24 February 2006.

Immunocytochemistry/ Immunofluorescence - Rad50 antibody [13B3/2C6] (ab89)

ab89 staining Rad50 in HPV transfected Hela cells by Immunocytochemistry/ Immunofluorescence. Cells were washed twice with phosphate-buffered saline (PBS), permeabilized with 0.5% Triton X-100 in CSK buffer (10 mM Hepes-KOH, pH 7.4; 300 mM sucrose; 100 mM NaCl; 3 mM KCl) for 2 min on ice, and fixed with 4% paraformaldehyde. Fixed cells were treated with 0.5% Triton X-100 in PBS, followed by 3 PBS washes for 5 min each at RT. After blocking with 3% bovine serum albumin (BSA) in PBS at RT for 30 min, the cells were incubated with primary antibody in antibody-binding solution (3% BSA in PBS) at RT for 30 min. An Alexa Fluor® 488 conjugated anti mouse secondary was used.

Image from Kadaja M et. al., PLoS Pathog. 2009 April; 5(4): e1000397 (Fig 4).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Rad50 antibody [13B3/2C6] (ab89)

ab89 (1µg/ml) staining RAD50 in human placenta, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining.
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.

Flow Cytometry-Anti-Rad50 antibody [13B3/2C6](ab89)

Overlay histogram showing HeLa cells stained with ab89 (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 (ab89, 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.