All lanes : Anti-Caldesmon/CDM antibody [E89] (ab32330) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CALD1 knockout HeLa cell lysate
Lane 3 : NIH/3T3 cell lysate
Lane 4 : HEK-293 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 93 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab32330 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa.

ab32330 Anti-Caldesmon/CDM antibody [E89] was shown to specifically react with Caldesmon/CDM in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265026 (knockout cell lysate ab257375) was used. Wild-type and Caldesmon/CDM knockout samples were subjected to SDS-PAGE. ab32330 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

Immunocytochemistry/Immunofluorescence analysis of C6 (rat glioma) cells labelling Caldesmon/CDM with purified ab32330 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Nuclei couterstained with DAPI (blue). 

Secondary Only Control: PBS was used instead of the primary antibody as the negative control. 

ab32300, at a 1/250 dilution, staining human Caldesmon/CDM in uterus tissue by Immunohistochemistry, Paraffin embedded tissue

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Anti-Caldesmon/CDM antibody [E89] (ab32330) at 1/20000 dilution + NIH3T3 cell lysate

Predicted band size: 93 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

ab32330, at a 1/250 dilution, staining human Caldesmon/CDM in HeLa cells by Immunofluorescence

Overlay histogram showing HeLa cells stained with ab32330 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab32330, 1/20 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 significantly decreased signal in HeLa cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

Fluorescent immunohistochemical analysis of paraffin-embedded human normal kidney vessels using ab32330. Green-Caldesmon/CDM red-PI

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Fluorescent immunohistochemical analysis of paraffin-embedded human tissue using ab32330. Green-Caldesmon/CDM red-PI.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Fluorescent immunohistochemical analysis of paraffin-embedded human normal uterus tissue using ab32330. Green-Caldesmon/CDM red-PI.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Equilibrium disassociation constant (K_D)
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