Anti-CaMKII antibody [EPR6686(2)]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CaMKII antibody [EPR6686(2)] (AB134041)

Intracellular 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 and then permeabilized with 90% methanol for 30min. 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).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CaMKII antibody [EPR6686(2)] (AB134041)

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CaMKII antibody [EPR6686(2)] (AB134041)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CaMKII antibody [EPR6686(2)] (AB134041)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CaMKII antibody [EPR6686(2)] (AB134041)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CaMKII antibody [EPR6686(2)] (AB134041)

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).

• WB

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Western blot - Anti-CaMKII antibody [EPR6686(2)] (AB134041)

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.

All lanes:

Western blot - Anti-CaMKII antibody [EPR6686(2)] (ab134041) at 0.08 µg/mL

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

Rat brain lysates at 15 µg

Lane 3:

Mouse brain lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 54 kDa

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• WB

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Western blot - Anti-CaMKII antibody [EPR6686(2)] (AB134041)

All lanes:

Western blot - Anti-CaMKII antibody [EPR6686(2)] (ab134041) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

HeLa lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti- rabbit at 1/2000 dilution

Predicted band size: 54 kDa

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• WB

Lab

Western blot - Anti-CaMKII antibody [EPR6686(2)] (AB134041)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of HeLa whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab134041 (1/20,000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique.

ab134041 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected under these conditions. This data was generated as part of a structured antibody stability study supporting Abcam’s transition from cold‑chain to ambient shipping.

All lanes:

Western blot - Anti-CaMKII antibody [EPR6686(2)] (ab134041) at 1/20000 dilution

All lanes:

HeLa whole cell lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

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Exposure time: 20s