Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human clear cell cancer of kidney tissue sections labeling CaMKII with Purified ab134041 at 1:500 dilution (1.5 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0)ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylinwas used as a counterstain

All lanes : Anti-CaMKII antibody [EPR6686(2)] (ab134041) at 0.08 µg/ml (purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Rat brain lysates
Lane 3 : Mouse brain lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 54 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

This antibody might cross-react with family members based on the high immunogen homology. That might be the reason why doublet are detected.

Flow Cytometry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling CaMKII with purified ab134041 at 1:70 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

ab134041 staining CaMKII in Neuro-2A cells. The cells were differentiated with 20μM Trans-retinoic acid and serum starved (0.1%FBS/DMEM) for 48hours. They were then fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with purified ab134041 at a 5μg/ml concentration and ab78078, Mouse monoclonal [2G10] to beta III Tubulin, at 5μg/ml concentration, followed by a further incubation at room temperature for 1h with an anti-rabbit AlexaFluor® 488 (ab150081) at 2 μg/ml (shown in green) and an anti-mouse AlexaFluor® 594 (ab150120) at 2 μg/ml (shown in pseudocolor red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

 

Overlay histogram showing SH-SY5Y cells stained with ab134041 (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 followed by the antibody (unpurified ab134041, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-CaMKII antibody [EPR6686(2)] (ab134041) at 1/1000 dilution (unpurified)

Lane 1 : Human fetal brain lysate
Lane 2 : HeLa lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-

rabbit at 1/2000 dilution

Predicted band size: 54 kDa

Immunohistochemical analysis of paraffin-embedded Human brain tissue labelling CaMKII with unpurified ab134041 at 1/250 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunofluorescent analysis of U87-MG cells labelling CaMKII with unpurified ab134041 at 1/250 dilution.