Revision 7

#13987Store 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, IF-IC, FC-FP, ChIP, ChIP-seq, C&R

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

57-65

Source/Isotype:

Rabbit IgG

UniProt ID:

#P04198, #P01106

Entrez-Gene Id:

4613, 4609

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
Immunofluorescence (Immunocytochemistry) 1:1600
Flow Cytometry (Fixed/Permeabilized) 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.

For a carrier free (BSA and azide free) version of this product see product #83277.

Specificity / Sensitivity

c-Myc/N-Myc (D3N8F) Rabbit mAb recognizes endogenous levels of total c-Myc and N-Myc proteins.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to a central region within human c-Myc protein.

Background

Members of the Myc/Max/Mad network function as transcriptional regulators with roles in various aspects of cell behavior, including proliferation, differentiation, and apoptosis (1). These proteins share a common basic-helix-loop-helix leucine zipper (bHLH-ZIP) motif required for dimerization and DNA-binding. Max was originally discovered based on its ability to associate with c-Myc and found to be required for the ability of Myc to bind DNA and activate transcription (2). Subsequently, Max has been viewed as a central component of the transcriptional network, forming homodimers as well as heterodimers with other members of the Myc and Mad families (1). The association between Max and either Myc or Mad can have opposing effects on transcriptional regulation and cell behavior (1). The Mad family consists of four related proteins; Mad1, Mad2 (Mxi1), Mad3, and Mad4, and the more distantly related members of the bHLH-ZIP family, Mnt and Mga. Like Myc, the Mad proteins are tightly regulated with short half-lives. In general, Mad family members interfere with Myc-mediated processes, such as proliferation, transformation, and prevention of apoptosis by inhibiting transcription (3,4).

  1. Baudino, T.A. and Cleveland, J.L. (2001) Mol Cell Biol 21, 691-702.
  2. Blackwood, E.M. and Eisenman, R.N. (1991) Science 251, 1211-7.
  3. Henriksson, M. and Lüscher, B. (1996) Adv Cancer Res 68, 109-82.
  4. Grandori, C. et al. (2000) Annu Rev Cell Dev Biol 16, 653-99.

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 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.
SignalSilence is a registered trademark of Cell Signaling Technology, Inc.
SimpleChIP 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.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 7
#13987

c-Myc/N-Myc (D3N8F) Rabbit mAb

Western Blotting Image 1: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using c-Myc/N-Myc (D3N8F) Rabbit mAb.
Western Blotting Image 2: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
Western blot analysis of extracts from HT-29 cells, untreated (-) or treated with JQ1 (1 μM; 72 hr; +), using c-Myc/N-Myc (D3N8F) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, the BET bromodomain inhibitor JQ1 inhibits c-Myc expression.
Western Blotting Image 3: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
Western blot analysis of extracts from K562 [c-Myc (+) / N-Myc (-)] and IMR-32 [c-Myc (-) / N-Myc (+)] cell lines using cMyc/N-Myc (D3N8F) Rabbit mAb (left), N-Myc (D1V2A) Rabbit mAb #84406 (middle), or β-Actin (13E5) Rabbit mAb #4970 (right). 
Western Blotting Image 4: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® c-Myc siRNA I #6341 (+), using c-Myc/N-Myc (D3N8F) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The c-Myc/N-Myc (D3N8F) Rabbit mAb confirms silencing of c-Myc expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
Immunofluorescence Image 1: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HT-29 cells, untreated (left) or treated with bromodomain inhibitor JQ1 (1 μM, 72 hr; right), using c-Myc/N-Myc (D3N8F) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red).
Flow Cytometry Image 1: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
Flow cytometric analysis of HT29 cells using c-Myc/N-Myc (D3N8F) Rabbit mAb, treated with JQ1 (1uM, 72 hours at 37C) (blue) and untreated (green). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from Daudi cells and c-Myc/N-Myc (D3N8F) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across NPM1, a known target gene of c-Myc (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from Daudi cells and c-Myc/N-Myc (D3N8F) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 5 (upper), including NPM1 (lower), a known target gene of c-Myc (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from Daudi cells and either c-Myc/N-Myc (D3N8F) 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 ATF4 promoter primers, SimpleChIP® Human NPM1 Intron 1 Primers #4779, 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.
CUT and RUN Image 1: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
CUT&RUN was performed with Daudi cells and c-Myc/N-Myc (D3N8F) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across ABR, a known target gene of c-MYC/N-Myc (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
CUT&RUN was performed with Daudi cells and c-Myc/N-Myc (D3N8F) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 1 (upper), including ABR (lower), a known target gene of c-MYC/N-Myc (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: c-Myc/N-Myc (D3N8F) Rabbit mAb Expand Image
CUT&RUN was performed with Daudi cells and either c-Myc/N-Myc (D3N8F) 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 human HNRNPL intron 1 primers, human ABR intron 1 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.