Anti-Calmodulin 1/2/3 antibody [EP799Y] - C-terminal (ab45689)

ΔSRCR peripheral blood monocyte-derived macrophages (PMMs) still function as hemoglobin-haptoglobin (Hb-Hp) scavengers.

Porcine PMMs were incubated for 24 hours in the presence of 100 μg/mol Hb-Hp then lysed with reducing SDS sample buffer and HO-1 protein expression analyzed by western blot.

For full details please see paper.

Blocking and Diluting buffer: 5% NFDM/TBST

ab45689 immunoprecipitating Calmodulin. 10 µg of cell lysate was incubated with primary antibody at a dilution of 1/20 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/10000.

Lane 1: Human skeletal muscle lysate (10 µg)
Lane 2: Human skeletal muscle lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab45689 in human skeletal muscle lysate

ab45689 staining Calmodulin in human testis tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/2000. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control: PBS in place of primary antibody (inset).

ab45689 at a 1:250 dilution staining Calmodulin in human urinary bladder carcinoma tissue by immunohistochemistry in paraffin embedded tissue.

Blocking and diluting buffer: 5% NFDM/TBST

ab45689 staining Calmodulin in mouse brain tissue sections by Immunohistochemistry (IHC-Fr - frozen sections).

Tissue was fixed with acetone and blocked with 10% serum for 60 minutes at 4°C. Samples were incubated with primary antibody (1/100 in PBS + 0.1% Triton) for 12 hours at 4°C. An Alexa Fluor^® 555-conjugated Goat anti-rabbit IgG polyclonal (1/400) was used as the secondary antibody.

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Overlay histogram showing MCF7 (Human breast adenocarcinoma cell line) cells stained with ab45689 (red line).

The cells were fixed with 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 (ab45689, 1/50 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 monoclonal IgG (0.5µg/1x10⁶ cells) used under the same conditions.

Acquisition of >5,000 events was performed.

This antibody gave a decreased signal in MCF7 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

ab45689 staining Calmodulin in NIH/3T3 (Mouse embryo fibroblast cell line) cells by flow cytometry.

Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabeled control: Cell without incubation with primary antibody and secondary antibody (Blue)