Anti-Dnmt3a antibody [64B1446] - ChIP Grade (ab13888)

Western blot - Dnmt3a antibody [64B1446] - ChIP Grade (ab13888)

Western blot analysis for Dnmt3a using ab13888 at 2 ug/ml against 10 µg of 293 cell lysate transfected with either mouse Dnmt3a (lane A) or mouse Dnmt3b (lane B).

Western blot analysis for Dnmt3a using ab13888 at 2 ug/ml against 10 µg of 293 cell lysate transfected with either mouse Dnmt3a (lane A) or mouse Dnmt3b (lane B).

Immunocytochemistry - Dnmt3a antibody [64B1446] - ChIP Grade (ab13888)

Immuno-staining of Dnmt3a using ab13888 at 5 ug/ml dilution on cultured HeLa cells. The nucleic staining is shown in most of the cells.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Dnmt3a antibody [64B1446] - ChIP Grade (ab13888)

ab13888 (2µg/ml) staining Dnmt3a in human testis using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining in the seminal vesicles.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 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.

Immunocytochemistry/ Immunofluorescence - Dnmt3a antibody [64B1446] - ChIP Grade (ab13888)

ICC/IF image of ab13888 stainedHepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab13888, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Flow Cytometry-Anti-Dnmt3a antibody [64B1446] - ChIP Grade(ab13888)

Overlay histogram showing HEK293 cells stained with ab13888 (red line). The cells were fixed with 4% paraformaldehyde (10 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. The cells were then incubated with the antibody (ab138889, 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 HEK293 cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.