Anti-SERCA2 ATPase antibody

• WB

Supplier Data

Western blot - Anti-SERCA2 ATPase antibody (AB3625)

All lanes:

Western blot - Anti-SERCA2 ATPase antibody (ab3625) at 0.1 µg/mL

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 50 µg

Lane 2:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 50 µg

Lane 3:

NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 50 µg

Predicted band size: 115 kDa

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Exposure time: 30s

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody (AB3625)

ab3625 staining human heart. Staining is localised to the cytoplasm/sarcoplasm.
Left panel : with primary antibody at 1 ug/ml. Right panel : isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the DAKO 3-in-1 antigen retrieval buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SERCA2 ATPase antibody (AB3625)

ICC/IF image of ab3625 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3625, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• IP

Supplier Data

Immunoprecipitation - Anti-SERCA2 ATPase antibody (AB3625)

SERCA2 ATPase was immunoprecipitated from HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate (1 mg for IP, 20% of IP loaded) with ab3625 at 6 μg/mg lysate. Western blot was performed from the immunoprecipitate using ab3625 at 1 μg/ml.

Lane 1 : ab3625 IP in HEK-293T whole cell lysate.

Lane 3 : ab3625 IP in HEK-293T whole cell lysate.

Lane 2 : Control IgG IP in HEK-293T whole cell lysate.

Detection : Chemiluminescence with exposure time of 3 minutes.

All lanes:

Immunoprecipitation - Anti-SERCA2 ATPase antibody (ab3625)

Predicted band size: 115 kDa

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• WB

Supplier Data

Western blot - Anti-SERCA2 ATPase antibody (AB3625)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Anti-SERCA2 ATPase antibody (ab320749, Left) staining at 1/1000 dilution showed similar expression profile/ molecular weight with Anti-SERCA2 ATPase antibody (ab3625, Right) at 1/1000 dilution. 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 [RM1176] (<a href='/ko/products/primary-antibodies/serca2-atpase-antibody-rm1176-ab320749'>ab320749</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate boiled at 20 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate unboiled at 20 µg

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate boiled at 20 µg

Lane 4:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate unboiled at 20 µg

Lane 5:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate boiled at 20 µg

Lane 6:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate unboiled at 20 µg

Lane 7:

H9c2 (2-1) (rat cardiomyoblast) whole cell lysate boiled at 20 µg

Lane 8:

H9c2 (2-1) (rat cardiomyoblast) whole cell lysate unboiled at 20 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 1/100000 dilution

Observed band size: 115 kDa,140 kDa

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Exposure time: 26s

• WB

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Western blot - Anti-SERCA2 ATPase antibody (AB3625)

Sample : Homogenate from Pig (lanes 1 and 2) or Rabbit (lane 4) aorta or lysate from cultured Rat aortic smooth muscle cells (lane 3)

Affinity purified Rabbit anti-SERCA2 (ab3625) (lanes 2, 3 and IP for lane 4) or control (ctrl) monoclonal anti-SERCA2 (lanes 1 and WB for lane 4).

Dilutions : 1 : 1,000 (lanes 1, 2 and 4) or 1 : 2,500 (lane 3)

All lanes:

Western blot - Anti-SERCA2 ATPase antibody (ab3625)

Predicted band size: 115 kDa

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• WB

CiteAb

Western blot - Anti-SERCA2 ATPase antibody (AB3625)

Western Blotting using Anti-SERCA2 ATPase antibody, ab3625. Publication image from Wada, J. et al., 2016, Nat Commun, 27677409. Legend direct from paper.

Effects of ANGPTL2-KD on human cardiomyocytes.(a) ANGPTL2 protein levels in conditioned medium of human iPS-derived cardiomyocytes transfected with siRNA targeting ANGPTL2 (siANGPTL2) or control siRNA (siControl) (n=4 per group). Maintenance medium for iPS-derived cardiomyocytes (medium) served as a negative control. (b) Representative western blot of ANGPTL2, AKT and SERCA2A proteins (left) and quantification of SERCA2A protein levels (right) in cardiomyocytes transfected with siANGPTL2 or control siRNA. Experiments were performed at least three times. Hsc70 served as a loading control. Levels in control siRNA cells were set to 1. (c) Relative expression of energy-related genes in cardiomyocytes transfected with siANGPTL2 or control siRNA (n=6 per group). Levels in control cells were set to 1. Data are means±s.e.m. Statistical significance was determined by Student's t test. *P<0.05, **P<0.01, †P<0.001 between groups. (d) Immunohistochemical staining using antibody against ANGPTL2 or isotype control IgG (N.C.) in serial sections of heart tissues from patients with non-chronic HF (non-CHF) (Case 1 : 77-year-old male with acute malignant lymphoma; Case 2 : 82-year-old female with carcinomatous peritonitis) or with chronic HF (CHF) (Case 1 : 63-year-old male; Case 2 : 38-year-old female). Middle panels show magnified images of boxed regions in upper panels. Scale bars, 50 µm. (e) Immunofluorescent staining ofαMHC (red; upper), CD31 (an endothelial cell marker) (red; middle), PERIOSTIN (a marker of cardiac fibroblasts) (red; lower) and ANGPTL2 (green) in heart tissues from a patient with CHF (Case 3 : 85-year-old female). Magnified images of areas in squares are shown at right. Scale bars, 50 µm.

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• WB

CiteAb

Western blot - Anti-SERCA2 ATPase antibody (AB3625)

Western Blotting using Anti-SERCA2 ATPase antibody, ab3625. Publication image from Wada, J. et al., 2016, Nat Commun, 27677409. Legend direct from paper.

ANGPTL2 down-regulation in heart blocks progression to HF.(a–c) Representative immunoblot and quantitation of ANGPTL2 (a), relative levels of PGC-1α and PPARα transcripts (b) and levels of AKT and SERCA2a proteins (c) in hearts of WT mice 2 weeks after intravenous injection of 1 x 1010 or 3 x 1010 vg per mouse of AAV6-shAngptl2-A or -B relative to uninjected controls. Values from uninjected mice without were set to 1. n=4 per group. (d) Time line for AAV6-shAngptl2-B treatment and echocardiography analysis in the TAC model, 8–10 per group. (e) Representative western blot and quantitation of ANGPTL2 in TAC-induced hypertrophied heart, 4 weeks after injection of 3 x 1010 vg per mouse of AAV6-shScramble or 1 x 1010 or 3 x 1010 vg per mouse of AAV6-shAngptl2-B. (f–h) Comparison of indicated parameters among mice injected with 3 x 1010 vg per mouse of AAV6-shScramble or 1 x 1010 or 3 x 1010 vg per mouse of AAV6-shAngptl2-B. (i) Representative M-mode echocardiography recordings (upper), HE-stained sections of heart mid-portion (middle, Scale bar, 1 mm) and gross appearance of whole heart (bottom) in sham animals, and in TAC animals 4 weeks after injection of AAV6-shScramble or AAV6-shAngptl2-B (1 x 1010 or 3 x 1010 vg per mouse). Ruler tick marks indicate 1 mm. (j) HW/BW ratios of animals in h. (k) Relative expression of genes associated with HF. Control values from sham surgeries were set to 1 (e,k). Hsc70 served as a loading control. Data are means±s.e.m. Statistical significance was determined by one-way (a–c,e,g,h,j,k) or two-way (f) ANOVA. *P<0.05, **P<0.01, †P<0.001.

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