Description
The retinoblastoma gene product (Rb) is well known as a tumor suppressor and is either absent or mutated in many human tumors. Retrovirus-mediated gene transfer of the wild-type Rb gene into several Rb mutant neoplastic cell lines suppresses their tumorgenicity. Rb is a 110-kDa nuclear phosphoprotein that undergoes differential phosphorylation during the cell cycle. During G1 phase, Rb is predominantly in a hypophosphorylated state. It becomes increasingly phosphorylated throughout the cell cycle until late mitosis, when substantial dephosphorylation occurs. Hypophosphorylated Rb interacts with a number of cellular proteins including the E2F transcription factor, several cyclins, RBP-1, RBP-2, c-Abl, c-myc, N-myc, and p46. Phosphorylation of Rb at various sites, by Cyclin-dependent protein kinases, inhibits the binding of Rb to these proteins. Rb is thought to mediate its effects, in part, via the repression of genes required for proliferation. For example, Rb is specifically recruited to promoters containing E2F sites and actively represses E2F mediated transcription. Rb also stimulates the activity of other transcription factors, although the mechanisms are less clearly defined. Thus, Rb appears to regulate transcription in its aim to control cell growth. The J112-906 monoclonal antibody recognizes Rb phosphorylated at serines 807 and 811 (pS807/pS811), which regulate c-Abl binding and cell cycle progression. The orthologous phosphorylation sites in mouse and rat Rb are pS800/pS804 and pS778/pS782, respectively.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed.
1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
4. Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
5. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
6. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
7. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
