All lanes : Anti-CaMKII delta antibody [EPR13095] (ab181052) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CAMK2D knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 56 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab181052 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab181052 was shown to react with CaM-kinase II  in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab267322 (knockout cell lysate ab257376) was used. Wild-type HEK-293T and CAMK2D knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab181052 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human thyroid carcinoma tissue sections labeling CaMKII delta with Purified ab181052 at 1:100 dilution (1.82 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: CaMKII delta knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: A431 cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab181052 observed at 56 kDa. Red - loading control, ab8245, observed at 37 kDa.

Unpurified ab181052 was shown to recognize CaMKII delta when CaMKII delta knockout samples were used, along with additional cross-reactive bands. Wild-type and CaMKII delta knockout samples were subjected to SDS-PAGE. ab181052 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 respectively 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 h at room temperature before imaging.

Lane 1 : Anti-CaMKII delta antibody [EPR13095] (ab181052) at 1/1000 dilution
Lanes 2-4 : Anti-CaMKII delta antibody [EPR13095] (ab181052) at 1/1000 dilution (purified)

Lane 1 : HeLa ( Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg
Lane 2 : SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates at 20 µg
Lane 3 : Mouse heart lysates at 20 µg
Lane 4 : Rat spleen lysates at 20 µg

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

Predicted band size: 56 kDa

Blocking and diluting buffer : 5% NFDM/TBST

All lanes : Anti-CaMKII delta antibody [EPR13095] (ab181052) at 1/2000 dilution (unpurified)

Lane 1 : SW480 whole cell lysate
Lane 2 : A431 whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 56 kDa

Blocking/ Dilution buffer: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cardiac muscle tissue sections labeling CaMKII delta with Purified ab181052 at 1:100 dilution (1.82 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemical analysis of paraffin embedded Human skeletal muscle tissue sections labeling CaMKII delta using unpurified ab181052 at a 1/100 dilution. A ready to use HRP Polymer for Rabbit IgG was used as the secondary. Hematoxylin counterstain. Heat mediated antigen retrieval was performed citrate buffer pH 6 before commencing with IHC staining protocol.