Anti-Creatine kinase B type antibody [EPR3927]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Creatine kinase B type antibody [EPR3927] (AB92452)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Creatine kinase B type with purified ab92452 at 1 : 50 dilution (2.0 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Creatine kinase B type antibody [EPR3927] (AB92452)

Immunofluorescence analysis of HeLa cells labelled with ab92452 (unpurified) at 1/100 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Creatine kinase B type antibody [EPR3927] (AB92452)

Overlay histogram showing HeLa cells stained with ab92452 (unpurified) (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 (ab92452, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Creatine kinase B type antibody [EPR3927] (AB92452)

Paraffin embedded Human glioma tissue labelled with ab92452 (unpurified) at 1/100 dilution.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Creatine kinase B type antibody [EPR3927] (AB92452)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Creatine kinase B type with purified ab92452 at 1/20 dilution (5 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Creatine kinase B type antibody [EPR3927] (AB92452)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human astrocytoma tissue sections labeling Creatine kinase B type with purified ab92452 at 1/1000 dilution (0.101 μg/ml). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

Lab

Western blot - Anti-Creatine kinase B type antibody [EPR3927] (AB92452)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : Creatine kinase B knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : SH-SY5Y cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab92452 observed at 45 kDa. Red - loading control, ab18058, observed at 2X kDa.

ab92452 (unpurified) was shown to specifically react with Creatine kinase B when ProteinX knockout samples were used. Wild-type and Creatine kinase B knockout samples were subjected to SDS-PAGE. ab92452 and ab18058 (loading control to vinculin) were both diluted 1/10 000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Creatine kinase B type antibody [EPR3927] (ab92452)

Predicted band size: 43 kDa

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• WB

Lab

Western blot - Anti-Creatine kinase B type antibody [EPR3927] (AB92452)

All lanes:

Western blot - Anti-Creatine kinase B type antibody [EPR3927] (ab92452) at 1/10000 dilution

All lanes:

Y79 (Human retinoblastoma retinoblastoma) whole cell lysates at 15 µg

Secondary

All lanes:

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

Predicted band size: 43 kDa

Observed band size: 43 kDa

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• WB

Lab

Western blot - Anti-Creatine kinase B type antibody [EPR3927] (AB92452)

All lanes:

Western blot - Anti-Creatine kinase B type antibody [EPR3927] (ab92452) at 1/10000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

Mouse cerebellum lysates at 20 µg

Lane 3:

Mouse brain lysates at 20 µg

Lane 4:

C6 (Rat glial tumor glial cell) whole cell lysates at 20 µg

Lane 5:

Rat brain lysates at 20 µg

Secondary

All lanes:

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

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

• WB

Unknown

Western blot - Anti-Creatine kinase B type antibody [EPR3927] (AB92452)

All lanes:

Western blot - Anti-Creatine kinase B type antibody [EPR3927] (ab92452) at 1/10000 dilution

Lane 1:

SHSY-5Y cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

Y-79 cell lysate at 10 µg

Lane 4:

SW840 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 43 kDa

false