Recombinant Anti-Cyclin D1 antibody [EP272Y] - BSA and Azide free (ab227561)

This WB data was generated using the same anti-Cyclin D1 antibody clone, EP272Y, in a different buffer formulation (cat# ab40754).

Lanes:
Lane 1: Empty
Lane 2: Wild type HAP1 whole cell lysate (20 µg)
Lane 3: Cyclin D1 knockout HAP1 whole cell lysate (20 µg)
Lane 4: A431 whole cell lysate (20 µg)

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 recognize Cyclin D1 when HAP1 knockout samples were used, along with additional cross-reactive bands. 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 1000 dilution and 1/10000 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 1/10000 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40754).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40754).

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

This IHC data was generated using the same anti-Cyclin D1 antibody clone, EP272Y, in a different buffer formulation (cat# ab40754).

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.