The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 33 kDa (predicted molecular weight: 33 kDa).
Ser/Thr-kinase component of cyclin D-CDK4 (DC) complexes that phosphorylate and inhibit members of the retinoblastoma (RB) protein family including RB1 and regulate the cell-cycle during G(1)/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complexes and the subsequent transcription of E2F target genes which are responsible for the progression through the G(1) phase. Hypophosphorylates RB1 in early G(1) phase. Cyclin D-CDK4 complexes are major integrators of various mitogenenic and antimitogenic signals. Also phosphorylates SMAD3 in a cell-cycle-dependent manner and represses its transcriptional activity. Component of the ternary complex, cyclin D/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex.
Involvement in disease
Defects in CDK4 are a cause of susceptibility to cutaneous malignant melanoma type 3 (CMM3) [MIM:609048]. Malignant melanoma is a malignant neoplasm of melanocytes, arising de novo or from a pre-existing benign nevus, which occurs most often in the skin but also may involve other sites.
Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. CDC2/CDKX subfamily. Contains 1 protein kinase domain.
Phosphorylation at Thr-172 is required for enzymatic activity. Phosphorylated, in vitro, at this site by CCNH-CDK7, but, in vivo, appears to be phosphorylated by a proline-directed kinase. In the cyclin D-CDK4-CDKN1B complex, this phosphorylation and consequent CDK4 enzyme activity, is dependent on the tyrosine phosphorylation state of CDKN1B. Thus, in proliferating cells, CDK4 within the complex is phosphorylated on Thr-172 in the T-loop. In resting cells, phosphorylation on Thr-172 is prevented by the non-tyrosine-phosphorylated form of CDKN1B.
Cytoplasm. Nucleus. Membrane. Cytoplasmic when non-complexed. Forms a cyclin D-CDK4 complex in the cytoplasm as cells progress through G(1) phase. The complex accumulates on the nuclear membrane and enters the nucleus on transition from G(1) to S phase. Also present in nucleoli and heterochromatin lumps. Colocalizes with RB1 after release into the nucleus.
Lanes 1: Wild-type HAP1 cell lysate (20 µg) Lanes 2: CDK4 knockout HAP1 cell lysate (20 µg) Lanes 1 and 2: Merged (red and green) signal. Green signal from target – ab95255 observed at 34 kDa. Red signal from loading control – ab8226 observed at 42 kDa
ab95255 was shown to react with CDK4 when CDK4 knockout samples were used, along with additional cross-reactive bands. Wild-type and CDK4 knockout samples were subjected to SDS-PAGE. ab95255 and ab8226 (loading control to beta actin) were diluted 1 µg/mL and 1/2000 respectively and incubated overnight at 4ºC. 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 1/10 000 dilution for 1 h at room temperature before imaging.
Western blot - Cdk4 antibody (ab95255)
Anti-Cdk4 antibody (ab95255) at 1 µg/ml + MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg
Secondary Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 33 kDa Observed band size : 33 kDa Additional bands at : 110 kDa,16 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 20 minutesA positive signal was also observed within HeLa whole cell lysate.
Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.