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Purified Mouse Anti-Human Underphosphorylated Retinoblastoma Protein (RB)
Purified Mouse Anti-Human Underphosphorylated Retinoblastoma Protein (RB)
Selective binding of G99-549 to underphosphorylated Rb. MOLT-4 cells (ATCC CRL-1582, Human T lymphoblast) were labeled with [32P]-orthophosphate and immunoprecipitated with either clone G99-549 (lanes 1 & 3) or clone G3-245 (lanes 2 & 4). G99-549 (Cat. No. 554164) recognizes only underphosphorylated Rb whereas G3-245 (Cat. No. 554136) recognizes all species of Rb (highly phosphorylated, phosphorylated and under phosphorylated). Lanes 1 & 2 - When P32-labeled IP extracts are run on a gel and detected via autoradiography, the radioactive label is greatly reduced in the G99-549 (lane 1) compared to the G3-245 (lane 2) immunoprecipitates. Lanes 3 & 4 - A western blot of the same gel probing for total Rb (Clone G3-245) shows that underphosphorylated Rb is present in the G99-549 immunoprecipitates (lane 3) whereas underphosphorylated, phosphorylated, and highly phosphorylated forms are present in the G3-245 immunoprecipitates (lane 4). pRb, underphosphorylated Rb. ppRb, phosphorylated and highly phosphorylated forms of Rb.
Selective binding of G99-549 to underphosphorylated Rb. MOLT-4 cells (ATCC CRL-1582, Human T lymphoblast) were labeled with [32P]-orthophosphate and immunoprecipitated with either clone G99-549 (lanes 1 & 3) or clone G3-245 (lanes 2 & 4). G99-549 (Cat. No. 554164) recognizes only underphosphorylated Rb whereas G3-245 (Cat. No. 554136) recognizes all species of Rb (highly phosphorylated, phosphorylated and under phosphorylated). Lanes 1 & 2 - When P32-labeled IP extracts are run on a gel and detected via autoradiography, the radioactive label is greatly reduced in the G99-549 (lane 1) compared to the G3-245 (lane 2) immunoprecipitates. Lanes 3 & 4 - A western blot of the same gel probing for total Rb (Clone G3-245) shows that underphosphorylated Rb is present in the G99-549 immunoprecipitates (lane 3) whereas underphosphorylated, phosphorylated, and highly phosphorylated forms are present in the G3-245 immunoprecipitates (lane 4). pRb, underphosphorylated Rb. ppRb, phosphorylated and highly phosphorylated forms of Rb.
Product Details
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BD Pharmingen™
RB (a.a. 514-610)
Human (QC Testing), Mouse (Tested in Development)
Mouse IgG1
Recombinant full-length human Rb
Immunoprecipitation (Routinely Tested)
105 kDa
0.5 mg/ml
AB_395279
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at 4°C.

Recommended Assay Procedures

Applications include immunoprecipitation at 1-2 µg/one million cells. Rb migrates as multiple, closely-spaced bands between approximately 110-116 kDa when sized on denaturing polyacrylamide gels (i.e. by SDS/PAGE). The multiple 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 (at least 12 inches) gel. MOLT-4 human leukemia cells (ATCC CRL-1582) are suggested as a positive control.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  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. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
554164 Rev. 10
Antibody Details
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G99-549

The retinoblastoma gene encodes a nuclear phosphoprotein (Rb or p110Rb) 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, adenoviral 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. G99-549 recognizes only the fast migrating, underphosphorylated form of Rb protein. G99-549 cross-reacts with mouse underphosphorylated Rb. The epitope has been mapped to amino acids 514-610 (box A) of human Rb. Recombinant full-length human Rb was produced in the baculovirus expression vector system (BEVS), purified and used as immunogen.

554164 Rev. 10
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
554164 Rev.10
Citations & References
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Development References (6)

  1. Cordon-Cardo C, Richon VM. Expression of the retinoblastoma protein is regulated in normal human tissues. Am J Pathol. 1994; 144(3):500-510. (Biology). View Reference
  2. Dunaief JL, Strober BE, Guha S, et al. The retinoblastoma protein and BRG1 form a complex and cooperate to induce cell cycle arrest. Cell. 1994; 79(1):119-130. (Clone-specific: Western blot). View Reference
  3. 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
  4. 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
  5. 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
  6. Weinberg RA. Tumor suppressor genes. Science. 1991; 254(5035):1138-1146. (Biology). View Reference
View All (6) View Less
554164 Rev. 10

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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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.