All lanes : Anti-Caldesmon/CDM antibody [SP226] (ab183339) 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 - ab183339 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa.

ab183339 Anti-Caldesmon/CDM antibody [SP226] 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. ab183339 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.

 

 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat colon tissue sections labeling Caldesmon/CDM with ab183339 at 1/100 dilution (23.11 µg/ml). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Positive staining on smooth muscle in rat colon, performed on a Leica Biosystems BOND™ RX instrument.
The section was incubated with ab183339 for 10 mins at room temperature.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse colon tissue sections labeling Caldesmon/CDM with ab183339 at 1/100 dilution (23.11 µg/ml). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Positive staining on smooth muscle in mouse colon, performed on a Leica Biosystems BOND™ RX instrument.
The section was incubated with ab183339 for 10 mins at room temperature.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human colon tissue sections labeling Caldesmon/CDM with ab183339 at 1/100 dilution (23.11 µg/ml). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Positive staining on smooth muscle in human colon, performed on a Leica Biosystems BOND™ RX instrument.
The section was incubated with ab183339 for 10 mins at room temperature.

Anti-Caldesmon/CDM antibody [SP226] (ab183339) at 1/400 dilution + Smooth muscle cell lysate

Predicted band size: 93 kDa

Flow cytometry analysis of Hela (human cervix adenocarcinoma) labeling Caldesmon/CDM with purified ab183339 at 1/230 dilution (10.05 µg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlableled control - Unlabelled cells (blue).

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human colon tissue labeling Caldesmon/CDM with ab183339 at 1/100 dilution.

Flow cytometric analysis of HeLa cells labeling Caldesmon/CDM with ab183339 at 1/400 dilution (green) compared to a negative control Rabbit IgG (blue).