Immunohistochemistry analysis of Phospho-RB [pT826] showing staining in the nucleus of paraffin-embedded human colon carcinoma tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-RB [pT826] polyclonal antibody (44576G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
|Tested species reactivity||Human|
|Published species reactivity||Mouse, Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human Rb that contains threonine 826 (based on Swiss Protein database, accession number P06400). The sequence is conserved in mouse, rat and chicken.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/million cells|
|Immunohistochemistry (Paraffin) (IHC (P))||1:10-1:100|
|Western Blot (WB)||Assay Dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Rb is a tumor suppressor nuclear phosphoprotein capable of binding to DNA. It is phosphorylated on serine and threonine, but not on tyrosine residues. It forms a complex with SV40 large T antigen, adenovirus E1A, and human papilloma virus-16 E. Rb protein may act by regulating transcription and loss of its function leads to uncontrolled cell growth. Aberrations in the RB gene have been implicated in cancers of breast, colon, prostate, kidney, nasopharynx, and leukemia. Phosphorylation of Rb at S608 depends on CDK4.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
p53 promotes VEGF expression and angiogenesis in the absence of an intact p21-Rb pathway.
44-576G was used in western blot to report the dual role for p53 in regulating vascular endothelial growth factor during hypoxia.
|Farhang Ghahremani M,Goossens S,Nittner D,Bisteau X,Bartunkova S,Zwolinska A,Hulpiau P,Haigh K,Haenebalcke L,Drogat B,Jochemsen A,Roger PP,Marine JC,Haigh JJ||Cell death and differentiation (20:888)||2013|
Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb, p107, and p130.
44-576G was used in western blot to investigate the roles of pRb, p107, and p130 in the cellular response to oxidative stress.
|Cicchillitti L,Fasanaro P,Biglioli P,Capogrossi MC,Martelli F||The Journal of biological chemistry (278:19509)||2003|