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