Revision 6

#7389Store 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, W-S, IP, IF-IC, ChIP, ChIP-seq

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

300

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q92793

Entrez-Gene Id:

1387

Product Information

Product Usage Information

For optimal ChIP 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.

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:50 - 1:250
Immunoprecipitation 1:200
Immunofluorescence (Immunocytochemistry) 1:100 - 1:200
Chromatin IP 1:50
Chromatin IP-seq 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

CBP (D6C5) Rabbit mAb recognizes endogenous levels of total CBP protein. This antibody does not cross-react with p300 protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a recombinant protein specific to the amino terminus of human CBP protein. The epitope has been mapped to residues surrounding Ser235.

Background

CBP (CREB-binding protein) and p300 are highly conserved and functionally related transcriptional co-activators that associate with transcriptional regulators and signaling molecules, integrating multiple signal transduction pathways with the transcriptional machinery (1,2). CBP/p300 also contain histone acetyltransferase (HAT) activity, allowing them to acetylate histones and other proteins (2). Phosphorylation of p300 at Ser89 by PKC represses its transcriptional activity, and phosphorylation at the same site by AMPK disrupts the association of p300 with nuclear receptors (3,4). Ser1834 phosphorylation of p300 by Akt disrupts its association with C/EBPβ (5). Growth factors induce phosphorylation of CBP at Ser437, which is required for CBP recruitment to the transcription complex (6). CaM kinase IV phosphorylates CBP at Ser302, which is required for CBP-dependent transcriptional activation in the CNS (7). The role of acetylation of CBP/p300 is of particular interest (2,8). Acetylation of p300 at Lys1499 has been demonstrated to enhance its HAT activity and affect a wide variety of signaling events (9).

  1. Goodman, R.H. and Smolik, S. (2000) Genes Dev 14, 1553-77.
  2. Chan, H.M. and La Thangue, N.B. (2001) J. Cell Sci. 114, 2363-2373.
  3. Yuan, L.W. and Gambee, J.E. (2000) J. Biol. Chem. 275, 40946-40951.
  4. Yang, W. et al. (2001) J. Biol. Chem. 276, 38341-38344.
  5. Guo, S. et al. (2001) J. Biol. Chem. 276, 8516-8523.
  6. Zanger, K. et al. (2001) Mol. Cell 7, 551-558.
  7. Impey, S. et al. (2002) Neuron 34, 235-244.
  8. Yuan, L.W. and Giordano, A. (2002) Oncogene 21, 2253-2260.
  9. Thompson, P.R. et al. (2004) Nat. Struct. Mol. Biol. 11, 308-315.

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 W-S: Simple Western™ IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq

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.
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 6
#7389

CBP (D6C5) Rabbit mAb

Western Blotting Image 1: CBP (D6C5) Rabbit mAb Expand Image
Western blot analysis of extracts from control HEK293T cells (Lane 1) or HEK293T cells with a targeted mutation in the gene encoding CBP (Lane 2) using CBP (D6C5) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The change in CBP molecular weight in the mutated HEK293T cells confirms the specificity of the antibody for CBP.
Western Blotting Image 2: CBP (D6C5) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using CBP (D6C5) Rabbit mAb.
Western Blotting Image 1: CBP (D6C5) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (0.1 mg/mL) from 293T cells using CBP (D6C5) Rabbit mAb #7389. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66 - 440 kDa separation module.
Immunoprecipitation Image 1: CBP (D6C5) Rabbit mAb Expand Image
Immunoprecipitation of CBP from HeLa cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or CBP (D6C5) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using CBP (D6C5) Rabbit mAb.
Immunofluorescence Image 1: CBP (D6C5) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HeLa cells using CBP (D6C5) Rabbit mAb (green) and DyLight 554 Phalloidin #13054 (red).
Chromatin Immunoprecipitation Image 1: CBP (D6C5) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells treated with Forskolin #3828 (30 μM, 1h) and CBP (D6C5) Rabbit mAb, 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. The figure shows binding across NR4A3, a known target gene of CBP (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product datasheet.
Chromatin Immunoprecipitation Image 2: CBP (D6C5) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells treated with Forskolin #3828 (30 μM, 1h) and CBP (D6C5) Rabbit mAb, 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. The figure shows binding across chromosome 9 (upper), including NR4A3 (lower), a known target gene of CBP (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: CBP (D6C5) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells, treated with Forskolin #3828 (30 μM, 1h) and either CBP (D6C5) 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 human ALS2 exon 1 primers, SimpleChIP® Human NR4A3 Promoter Primers #4829, 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.