Store at –20°C Rb (4H1) Mouse mAb #9309 n Small 100 µl (10 western blots) Orders n 877-616-CELL (2355) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com n Large 300 µl (30 western blots) n Trial 20 µl (2 western blots) rev. 06/11/15 For Research Use Only. Not For Use In Diagnostic Procedures. Entrez-Gene ID #2932 UniProt ID #P49841 Applications Species Cross-Reactivity* Molecular Wt. Isotype W, IP, IHC-P, IF-IC, ChIP, F Endogenous H, Mk, Pg, B 110 kDa Mouse IgG2a** Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody. *Species cross-reactivity is determined by western blot. Background: The retinoblastoma tumor suppressor protein, Rb, regulates cell proliferation by controlling progression through the restriction point within the G1phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase and proteins with a conserved LXCXE motif (2–4). Cell cycle-dependent phosphorylation by CDK’s inhibits Rb target binding, thus allowing cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires first phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6–8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9). Specificity/Sensitivity: Rb (4H1) Mouse mAb detects endogenous levels of total Rb protein. The antibody does not cross-react with the Rb homologues p107 or p130, or with other proteins. **Anti-mouse secondary antibodies must be used to detect this antibody. Recommended Antibody Dilutions: Western blotting 1:2000 Immunoprecipitation1:100 Immunohistochemistry (Paraffin) 1:100 Unmasking buffer: Citrate Antibody diluent: SignalStain® Antibody Diluent #8112 Immunofluorescence (IF-IC)1:200 Chromatin IP 1:100 Flow Cytometry 1:400 Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing nuclear localization, using Rb (4H1) Mouse mAb. Source/Purification: Monoclonal antibody is produced by immunizing animals with a Rb-C fusion protein containing residues 701-928 of human Rb. kDa Retinoblastoma, Cell Cycle Checkpoint section Rb 105 Extracellular Signals 76 57 Cyclin D cdk4 Western blot analysis of extracts from COS-7 cells, untreated or hydroxyurea-treated (G1/S), using Rb (4H1) Mouse mAb. Confocal immunofluorescent image of SH-SY5Y cells using RB (4H1) Mouse mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Cyclin A cdk2 Ser608 Ser780 Ser795 Cyclin E cdk2 Rb Thr373 Ser249/252 Ser807/811 W—Western Species Cross-Reactivity Key: IP—Immunoprecipitation H—human M—mouse Dg—dog Pg—pig Sc—S. cerevisiae Ce—C. elegans IHC—Immunohistochemistry R—rat Hr—Horse Hm—hamster ChIP—Chromatin Immunoprecipitation Mk—monkey All—all species expected Mi—mink C—chicken Tween® is a registered trademark of ICI Americas, Inc. IF—Immunofluorescence F—Flow cytometry Dm—D. melanogaster X—Xenopus Z—zebrafish Species enclosed in parentheses are predicted to react based on 100% homology. E-P—ELISA-Peptide B—bovine E2F DNA Synthesis S Restriction Point IMPORTANT: For western blots, incubate membrane with diluted antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight. + Rb G1 Applications Key: 0 Retinoblastoma, Cell Cycle Checkpoint Section x-x-xxxx Rb.eps Cont. G1/S 165 ® 2015 Cell Signaling Technology, Inc. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. For application specific protocols please see the web page for this product at www.cellsignal.com. Please visit www.cellsignal.com for a complete listing of recommended companion products. Background References: (1) Sherr, C.J. (1996) Science 274, 1672–1677. (2) Nevins, J.R. et al. (1992) Science 258, 424–429. 3 10 Rb 102 (3) Welch, P.J. and Wang, J.Y. (1993) Cell 75, 779–790. (4) Hu, Q.J. et al. (1990) EMBO J. 9, 1147–1155. 101 (5) Knudsen, E.S. and Wang, J.Y. (1997) Mol. Cell. Biol. 17, 5771–5783. 100 (6) Lundberg, A.S. and Weinberg, R.A. (1998) Mol. Cell. Biol. 18, 753–761. 0 Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Rb (4H1) Mouse mAb. 1023 DNA (PI) Flow cytometric analysis of Jurkat cells using Rb (4H1) Mouse mAb versus propidium iodide (DNA content). The box indicates Rb positive cells. (7) Connell-Crowley, L. et al. (1997) Mol. Cell. Biol. 8, 287–301. (8) Kitagawa, M. et al. (1996) EMBO J. 15, 7060–7069. (9) Geng, Y. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 194–199. Signal relative to input Rb (4H1) Mouse mAb #9309 Normal Rabbit IgG #2729 0.0045 0.0040 0.0035 0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0 Timeless DHFR α Satellite ® 2015 Cell Signaling Technology, Inc. Chromatin immunoprecipitations were performed with crosslinked chromatin from 4 x 106 Raji cells and either 5 μl of Rb (4H1) Mouse mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using using SimpleChIP® Human Timeless Intron 1 Primers #7001, human DHFR promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one. Orders n 877-616-CELL (2355) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com
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