Revision 3

#76266Store 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, ChIP

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

62

Source/Isotype:

Rabbit IgG

UniProt ID:

#P12524

Entrez-Gene Id:

4610

Product Information

Product Usage Information

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

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:200
Chromatin IP 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 -20ºC. Do not aliquot the antibody.

Specificity / Sensitivity

L-Myc (E3M5P) Rabbit mAb recognizes endogenous levels of total L-Myc protein. Cross-reactivity was not observed with other family members.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human L-Myc protein. The epitope has been mapped to residues surrounding Thr190.

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).

The Myc family is comprised of c-Myc, N-Myc, and L-Myc with often distinct patterns of expression during development as well as cancer (5). Amplification of each of the family members in cancer is frequently mutually exclusive with c-Myc being the most widely studied and most commonly amplified. N-Myc amplification, on the other hand, is found predominantly in neuroblastomas, and L-Myc amplification has been described in small cell lung cancer (SCLC) (6). Amplification of L-Myc in SCLC may result from chromosomal rearrangement between the rlf and MYCL genes (7). L-Myc expression by dendritic cells may be required for optimal T cell priming (8).

  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.
  5. Nesbit, C.E. et al. (1999) Oncogene 18, 3004-16.
  6. Nau, M.M. et al. Nature 318, 69-73.
  7. Mäkelä, T.P. et al. (1991) EMBO J 10, 1331-5.
  8. KC, W. et al. (2014) Nature 507, 243-7.

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 ChIP: Chromatin IP

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.
SimpleChIP is a registered 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.

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Revision 3
#76266

L-Myc (E3M5P) Rabbit mAb

Western Blotting Image 1: L-Myc (E3M5P) Rabbit mAb Expand Image
Western blot analysis of various cell lines using L-Myc (E3M5P) Rabbit mAb (upper), c-Myc (E5Q6W) Rabbit mAb #18583 (upper-middle), N-Myc (D4B2Y) Rabbit mAb #51705 (lower-middle), and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The cell type specificity for each of the Myc family members is predicted from RNAseq data and confirms the specificity of the antibodies. NCI-H1963 cells express both wild-type and an RLF-L-Myc fusion protein resulting from intrachromosomal rearrangement.
Western Blotting Image 2: L-Myc (E3M5P) Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human L-Myc protein (hMYCL-Myc/DDK; +), using L-Myc (E3M5P) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Immunoprecipitation Image 1: L-Myc (E3M5P) Rabbit mAb Expand Image
Immunoprecipitation of L-Myc protein from NCI-H209 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is L-Myc (E3M5P) Rabbit mAb. Western blot analysis was performed using L-Myc (E3M5P) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: L-Myc (E3M5P) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCI-H209 cells and either L-Myc (E3M5P) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using human ATF4 promoter primers, human NPM-1 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.