Revision 4

#5694Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IF-IC, FC-FP, ChIP, ChIP-seq, C&R

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

41

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q99496

Entrez-Gene Id:

6045

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:200
Immunofluorescence (Immunocytochemistry) 1:250 - 1:1000
Flow Cytometry (Fixed/Permeabilized) 1:100 - 1:400
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 1:50

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.

Specificity / Sensitivity

RING1B (D22F2) XP® Rabbit mAb recognizes endogenous levels of total RING1B protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the full-length human RING1B protein.

Background

The polycomb group (PcG) proteins contribute 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). Methylation of Lys27 facilitates the recruitment of the second complex, PRC1, which ubiquitinates histone H2A on Lys119 (6). PRC1 is composed of Bmi1 and RING1A (also RING1 or RNF1), both of which act to enhance the E3 ubiquitin ligase activity of an additional catalytic subunit RING1B (also RING2 or RNF2) (7). PcG proteins play an important role in the regulation of cell proliferation and senescence through repression of the p16 INK4A and p19 ARF genes and are required for maintenance of adult hematopoietic and neural stem cells, as well as embryonic stem cells (3,4,8-10).

  1. Boyer, L.A. et al. (2006) Nature 441, 349-53.
  2. Lee, T.I. et al. (2006) Cell 125, 301-13.
  3. Park, I.K. et al. (2003) Nature 423, 302-5.
  4. Molofsky, A.V. et al. (2003) Nature 425, 962-7.
  5. Cao, R. and Zhang, Y. (2004) Mol Cell 15, 57-67.
  6. Wang, H. et al. (2004) Nature 431, 873-8.
  7. Cao, R. et al. (2005) Mol Cell 20, 845-54.
  8. Molofsky, A.V. et al. (2005) Genes Dev 19, 1432-7.
  9. Jacobs, J.J. et al. (1999) Nature 397, 164-8.
  10. Jacobs, J.J. et al. (1999) Genes Dev 13, 2678-90.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 4
#5694

RING1B (D22F2) XP® Rabbit mAb

Western Blotting Image 1: RING1B (D22F2) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using RING1B (D22F2) XP® Rabbit mAb.
No image available
Immunofluorescence Image 1: RING1B (D22F2) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HeLa cells using RING1B (D22F2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow Cytometry Image 1: RING1B (D22F2) XP® Rabbit mAb Expand Image
Flow cytometric analysis of HeLa cells using RING1B (D22F2) XP® Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: RING1B (D22F2) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either RING1B (D22F2) XP® Rabbit mAb or Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. RING1B and H3K27me3 are known to associate with each other on chromatin. The figure shows binding of both RING1B and H3K27me3 across HOXD genes, which are known target genes of RING1B (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product datasheet.
Chromatin Immunoprecipitation Image 2: RING1B (D22F2) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either RING1B (D22F2) XP® Rabbit mAb or Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. RING1B and H3K27me3 are known to associate with each other on chromatin. The figure shows binding of both RING1B and H3K27me3 across HOXA genes (upper) and HOXD genes (lower), which are known target genes of RING1B (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: RING1B (D22F2) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either RING1B (D22F2) XP® Rabbit mAb or 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).
CUT and RUN Image 1: RING1B (D22F2) XP® Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and either RING1B (D22F2) XP® Rabbit mAb or Bmi1 (D42B3) Rabbit mAb #5856, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. RING1B and Bmi1 are both PRC1 components. The figure shows binding across COMMD3 gene.
CUT and RUN Image 2: RING1B (D22F2) XP® Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and either RING1B (D22F2) XP® Rabbit mAb or Bmi1 (D42B3) Rabbit mAb #5856, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. RING1B and Bmi1 are both PRC1 components. The figures show binding across HOXD (upper) and COMMD3 (lower) genes.
CUT and RUN Image 3: RING1B (D22F2) XP® Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and either RING1B (D22F2) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. 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, which is equivalent to one.