Download Bmi1 (D42B3) Rabbit mAb - Cell Signaling Technology

Store at -20°C
Bmi1 (D42B3) Rabbit mAb
#5856
Orders n 877-616-CELL (2355)
[email protected]
Support n 877-678-TECH (8324)
[email protected]
Web n www.cellsignal.com
rev. 03/10/17
For Research Use Only. Not For Use In Diagnostic Procedures.
Entrez-Gene ID #648
UniProt ID #P35226
Applications
W, IP, IF-IC, ChIP, ChIP-seq
Endogenous
Species Cross-Reactivity*
H, M, R, Mk
Isotype
Rabbit IgG**
Molecular Wt.
41, 43 kDa
Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM
NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C.
Do not aliquot the antibody.
® 2015 Cell Signaling Technology, Inc.
SimpleChIP and Cell Signaling Technology® are trademarks of Cell Signaling Technology, Inc.
Specificity/Sensitivity: Bmi1 (D42B3) Rabbit mAb
recognizes endogenous levels of total Bmi1 protein.
Source/Purification: Monoclonal antibody is produced
by immunizing animals with recombinant protein specific to
the carboxy terminus of human Bmi1 protein.
H/
3
H- T3
4IICO E
S7
**Anti-rabbit secondary antibodies must be used to
detect this antibody.
NI
He
kDa
Recommended Antibody Dilutions:
Western blotting
1:1000
Immunoprecipitation1:100
Immunofluorescence (IF-IC)1:100
Chromatin IP
1:50
Chromatin IP-seq
1:50
200
140
100
80
60
50
Bmi1
40
For application specific protocols please see the web
page for this product at www.cellsignal.com.
30
20
Western blot analysis of extracts from various cell lines using
Bmi1 (D42B3) Rabbit mAb.
Please visit www.cellsignal.com for a complete listing
of recommended companion products.
Background References:
(1) Boyer, L.A. et al. (2006) Nature 441, 349-53.
(2) Lee, T.I. et al. (2006) Cell 125, 301-13.
Bmi1 (D42B3) Rabbit mAb #5856
Normal Rabbit IgG #2729
Signal relative to input
Background: The polycomb group (PcG) of proteins
contributes to the maintenance of cell identity, stem cell
self-renewal, cell cycle regulation, and oncogenesis by
maintaining the silenced state of genes that promote cell
lineage specification, cell death, and cell-cycle arrest
(1-4). PcG proteins exist in two complexes that cooperate
to maintain long-term gene silencing through epigenetic
chromatin modifications. The first complex, EED-EZH2, is
recruited to genes by DNA-binding transcription factors and
methylates histone H3 on Lys27. This histone methyltransferase activity requires the Ezh2, Eed, and Suz12
subunits of the complex (5). Histone H3 methylation at
Lys27 facilitates the recruitment of the second complex,
PRC1, which ubiquitinylates histone H2A on Lys119 (6).
Bmi1 is a component of the PRC1 complex, which together
with Ring1 strongly enhances the E3 ubiquitin ligase activity
of the Ring2 catalytic subunit (7). Bmi1 plays an important
role in the regulation of cell proliferation and senescence
through repression of the p16 INK4A and p19 ARF genes
and is required for maintenance of adult hematopoietic and
neural stem cells (3,4,8-10).
La
*Species cross-reactivity is determined by western blot.
(3) Park, I.K. et al. (2003) Nature 423, 302-5.
0.0030
(4) Molofsky, A.V. et al. (2003) Nature 425, 962-7.
0.0025
(5) Cao, R. and Zhang, Y. (2004) Mol Cell 15, 57-67.
0.0020
(6) Wang, H. et al. (2004) Nature 431, 873-8.
0.0015
(7) Cao, R. et al. (2005) Mol Cell 20, 845-54.
0.0010
(8) Molofsky, A.V. et al. (2005) Genes Dev 19, 1432-7.
0.0005
0
(9) Jacobs, J.J. et al. (1999) Nature 397, 164-8.
HoxA1
HoxA2
α Satellite
(10) J acobs, J.J. et al. (1999) Genes Dev 13, 2678-90.
Chromatin immunoprecipitations were performed with crosslinked chromatin from 4 x 106 NCCIT cells and either 10 µl of
Bmi1 (D42B3) Rabbit 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 SimpleChIP® Human HoxA1 Intron 1 Primers #7707,
SimpleChIP® Human HoxA2 Promoter Primers #5517, 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
(equivalent to one).
IMPORTANT: For western blots, incubate membrane
with diluted antibody in 5% w/v BSA, 1X TBS, 0.1%
Tween®20 at 4°C with gentle shaking, overnight.
Confocal immunofluorescent analysis of C2C12 cells using
BMI1 (D42B3) Rabbit mAb (green). Actin filaments were labeled
with DyLight™ 554 Phalloidin #13054 (red).
Applications Key:
W—Western
Species Cross-Reactivity Key:
IP—Immunoprecipitation
H—human
M—mouse
Dg—dog Pg—pig Sc—S. cerevisiae Ce—C. elegans
DyLight is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries.
Illumina is a registered trademark of Illumina, Inc.
NEBNext is a registered trademark of New England Biolabs, Inc.
Tween is a registered trademark of ICI Americas, Inc.
Ultra is a trademark of New England Biolabs, Inc.
IHC—Immunohistochemistry
R—rat
Hr—horse
Hm—hamster
ChIP—Chromatin Immunoprecipitation
Mk—monkey
Mi—mink
All—all species expected
C—chicken
IF—Immunofluorescence
F—Flow cytometry
Dm—D. melanogaster X—Xenopus
Z—zebrafish
E-P—ELISA-Peptide
B—bovine
Species enclosed in parentheses are predicted to react based on 100% homology.
26,800 kb
26,900 kb
27,000 kb
27,100 kb
27,200 kb
27,300 kb
HOXA-AS3 HOXA10 HOXA13
EVX1
27,400 kb
27,500 kb
Bmi1 (#5856)
[0-10]
RING1B (#5694)
[0-15]
SKAP2
HOXA1
176,900 kb
HOXA3
177,000 kb
177,100 kb
Bmi1 (#5856)
[0-15]
RING1B (#5694)
[0-25]
EVX2
HOXD12 HOXD10
MIR10B
HOXD3
HOXD1
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 10 NCCIT cells and either 10 µl of Bmi1 (D42B3) Rabbit mAb or 10 μl of RING1B (D22F2) XP® Rabbit mAb
#5694, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 5 ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for
Illumina®, and sequenced on the Illumina NextSeq. Bmi1 and RING1B are known to associate with each other on chromatin. The figure shows binding of both Bmi1 and RING1B across HOXA gene
cluster (upper) and HOXD gene cluster (lower), which are known target genes of Bmi1 and RING1B (see additional figure containing ChIP-qPCR data).
® 2015 Cell Signaling Technology, Inc.
6
Orders n 877-616-CELL (2355)
[email protected]
Support n 877-678-TECH (8324)
[email protected]
Web n www.cellsignal.com