All lanes : Anti-Calcium Pump PMCA4 ATPase antibody [JA9] (ab2783) at 1/500 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ATP2B4 knockout HeLa cell lysate
Lane 3 : SH-SY5Y cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Observed band size: 140 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab2783 observed at 140 kDa. Red - loading control, ab181602 observed at 37 kDa.

ab2783 Anti-Calcium Pump PMCA4 ATPase antibody [JA9] was shown to specifically react with Calcium Pump PMCA4 ATPase in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264720 (knockout cell lysate ab258321) was used. Wild-type and Calcium Pump PMCA4 ATPase knockout samples were subjected to SDS-PAGE. ab2783 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

 

IHC image of ab2783 staining in human cervical carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab2783, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Overlay histogram showing HeLa cells stained with ab2783 (red line). The cells were fixed with 80% methanol (5 min) and 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 (ab2783, 2μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. 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.
Please note that Abcam do not have any data for use of this antibody on non-fixed cells. We welcome any customer feedback.

Immunohistochemistry was performed on normal biopsies of deparaffinized Human brain tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing PMCA4 ATPase ab2783 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunolocalization of Calcium Pump PMCA4 ATPase in human corneal epithelium.

(A) Immunolocalization of PMCA4 (green) using ab2783 on cell membranes of all cells in each layer of human corneal epithelium.

(B) Immunolocalization of Calcium Sensing Receptor (red) using ab19347 on cell membranes of human corneal epithelium.

(C) Merged image.

Immunolocalization of Calcium Pump PMCA4 and Calcium Sensing Receptor in bovine corneal epithelium (bCE).

(A) Immunolocalization of Calcium Pump PMCA4 (green) using ab2783 on cell membranes of all cells in each layer of bCE.

(B) Immunolocalization of Calcium Sensing Receptor (red) using ab19347 on cell membranes of bCE mostly in wing- and squamous cell layers.

(C) Merged image.