Preparation And Storage
Recommended Assay Procedures
Application includes western blot analysis (1-2 µg/ml). Rb migrates as multiple closely-spaced bands between approximately 110-116 kDa when sized on denaturing polyacrylamide gels (i.e. by SDS-PAGE). The different bands represent different Rb phosphorylation states, the higher molecular weight bands are more highly phosphorylated than the lower molecular weight bands. The level of phosphorylation is cell cycle dependent, and may also be cell type dependent (not all forms are seen in all cell types that express Rb). Gel conditions influence the actual number of bands observed. In cases where optimal band separation is desired, use a 7 to 10% non-gradient long (≥ 12 inches) gel. MOLT-4 human leukemia cells (ATCC CRL-1582) are suggested as a positive control. Additional applications not routinely tested at BD Biosciences Pharmingen include immunoprecipitation (1-2 µg/ml) and electrophoretic mobility gel shift assays (EMSA).
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
The retinoblastoma gene encodes a nuclear phosphoprotein (Rb or p110[Rb]) which is expressed in most normal cells of vertebrates and acts as a tumor suppressor gene product. An underphosphorylated form of RB is mainly found in resting or fully differentiated cells, whereas the hyperphosphorylated form is present in proliferating cells. Only the underphosphorylated form of RB binds specifically to viral oncogenes such as SV40 large T, adenviral EIA and HPV-E7. This interaction may partially contribute to the transforming activity of these viral oncoproteins. Rb also interacts with several cyclins including A, D, and E as well as the transcriptional activator E2F. The importance of these interactions for the biological function of Rb is still being elucidated.
XZ55 recognizes an epitope located between amino acids 443-622 of human Rb. It cross-reacts with chicken, mouse and Xenopus Rb, and a putative quail Rb.A recombinant carboxy-terminal (amino acids 387-928) human Rb protein was used as immunogen.
Development References (6)
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Guilhot C, Benchaibi M, Flechon JE, Samarut J. The 12S adenoviral E1A protein immortalizes avian cells and interacts with the avian RB product. Oncogene. 1993; 8(3):619-624. (Clone-specific: Western blot). View Reference
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Hollingsworth RE Jr, Chen PL, Lee WH. Integration of cell cycle control with transcriptional regulation by the retinoblastoma protein. Curr Opin Cell Biol. 1993; 5(2):194-200. (Biology). View Reference
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Hu QJ, Bautista C, Edwards GM, Defeo-Jones D, Jones RE, Harlow E. Antibodies specific for the human retinoblastoma protein identify a family of related polypeptides. Mol Cell Biol. 1991; 11(11):5792-5799. (Clone-specific: Immunoprecipitation, Western blot). View Reference
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Livingston DM. Functional analysis of the retinoblastoma gene product and of RB-SV40 T antigen complexes. Cancer Surv. 1992; 12:153-160. (Biology). View Reference
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Riley DJ, Lee EY, Lee WH. The retinoblastoma protein: more than a tumor suppressor. Annu Rev Cell Biol. 1994; 10:1-29. (Biology). View Reference
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Shirodkar S, Ewen M, DeCaprio JA, Morgan J, Livingston DM, Chittenden T. The transcription factor E2F interacts with the retinoblastoma product and a p107-cyclin A complex in a cell cycle-regulated manner. Cell. 1992; 68(1):157-166. (Clone-specific: Gel shift). View Reference
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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
