All lanes : Anti-Cyclin D1 antibody [EP272Y] (ab40754) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CCND1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab40754 observed at 36 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.

 ab40754 was shown to react with Cyclin D1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255348 (knockout cell lysate ab263808) was used. Wild-type HeLa and CCND1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab40754 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 1000 dilution and a 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.

Immunohistochemical analysis of paraffin-embedded human kidney carcinoma using anti-Cyclin D1 RabMAb (ab40754).

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

All lanes : Anti-Cyclin D1 antibody [EP272Y] (ab40754) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CCND1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab40754 observed at 36 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab40754 was shown to react with Cyclin D1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261760 (knockout cell lysate ab256864) was used. Wild-type HeLa and CCND1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab40754 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 dilution and a 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.

All lanes : Anti-Cyclin D1 antibody [EP272Y] (ab40754) at 1/1000 dilution

Lane 2 : Wild-type HAP1 whole cell lysate
Lane 3 : Cyclin D1 knockout HAP1 whole cell lysate
Lane 4 : A431 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 33 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab40754 observed at 35 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab40754 was shown to specifically recognize Cyclin D1 when HAP1 in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when knockout samples were examined. Wild-type and HAP1 knockout samples were subjected to SDS-PAGE. ab40754 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

ab40754 staining cyclin D1 in MCF7 cells treated with KN-93 (water soluble) (ab120980), by ICC/IF. Decrease in cyclin D1 expression correlates with increased concentration of KN-93 (water soluble), as described in literature.
The cells were incubated at 37°C for 24 hours in media containing different concentrations of ab120980 (KN-93 (water soluble)) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab40754 (1/100 dilution) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A goat anti-rabbit DyLight 488 goat anti-rabbit secondary antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

Anti-Cyclin D1 antibody [EP272Y] (ab40754) at 1/2000 dilution + MCF-7 cell lysate at 10 µg

Predicted band size: 33 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma using anti-Cyclin D1 RabMAb (ab40754).

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