Anti-SERCA2 ATPase antibody [EPR9392]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [EPR9392] (AB150435)

Immunohistochemical analysis of paraffin embedded Human liver tissue labelling SERCA2 ATPase with unpurified ab150435 at 1/50.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SERCA2 ATPase antibody [EPR9392] (AB150435)

Immunocytochemistry/ Immunofluorescence analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling SERCA2 ATPase with Purified ab150435 at 1/100 dilution (10 μg/mL). Cells were fixed in 100% Methanol. 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-SERCA2 ATPase antibody [EPR9392] (AB150435)

Immunofluorescence analysis of HeLa cells labelling SERCA2 ATPase with unpurified ab150435 at 1/100.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [EPR9392] (AB150435)

Immunohistochemical analysis of paraffin embedded Human kidney tissue labelling SERCA2 ATPase with unpurified ab150435 at 1/50.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SERCA2 ATPase antibody [EPR9392] (AB150435)

Overlay histogram showing HepG2 cells stained with unpurified ab150435 (red line). The cells were fixed with 80% methanol (5 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 (ab150435, 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 HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SERCA2 ATPase antibody [EPR9392] (AB150435)

Intracellular Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling SERCA2 ATPase with Purified ab150435 at 1/1000 dilution (1 μg/mL) (Red). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. 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-SERCA2 ATPase antibody [EPR9392] (AB150435)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human lung carcinoma tissue sections labeling SERCA2 ATPase with purified ab150435 at 1/50 dilution (20 µg/mL). Perform heat mediated antigen retrieval 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. Hematoxylin was used as a counterstain.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [EPR9392] (AB150435)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human brain tissue sections labeling SERCA2 ATPase with purified ab150435 at 1/50 dilution (20 µg/mL). Perform heat mediated antigen retrieval 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. Hematoxylin was used as a counterstain.

• WB

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Western blot - Anti-SERCA2 ATPase antibody [EPR9392] (AB150435)

Blocking/Diluting buffer : 5% NFDM/TBST

Suggest to use non-boiled samples, as boiling process could cause membrane protein aggregates (PMID : 16023741 and PMID : 8670158).

All lanes:

Western blot - Anti-SERCA2 ATPase antibody [EPR9392] (ab150435) at 1/5000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates boiled at 20 µg

Lane 4:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates unboiled at 20 µg

Lane 5:

Mouse brain lysates boiled at 20 µg

Lane 6:

Mouse brain lysates unboiled at 20 µg

Lane 7:

Rat brain lysates boiled at 20 µg

Lane 8:

Rat brain lysates unboiled 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: 115 kDa

Observed band size: 115 kDa,140 kDa

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