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.

ICC/IF image of ab45689 stained MCF7 (Human breast adenocarcinoma cell line) cells.

The cells were fixed in 100% methanol for 5 minutes and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab45689, 1/1000 dilution) 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.

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 secondary antibody (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.

See Abreview

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)