Revision 5

#77087Store 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, C&R

REACTIVITY:

H Mk

SENSITIVITY:

Endogenous

MW (kDa):

185

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q8TEK3

Entrez-Gene Id:

84444

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
Chromatin IP 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

DOT1L (D1W4Z) Rabbit mAb recognizes endogenous levels of total DOT1L protein.

Species Reactivity:

Human, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro459 of human DOT1L protein.

Background

DOT1-like protein (DOT1L), also known as Lysine N-methyltransferase 4 (KMT4), is a histone methyltransferase that functions to mono-, di-, and tri-methylate histone H3 on lysine 79, a histone modification that is associated with active transcription and plays a role in DNA damage response, cell cycle regulation, and embryonic stem cell development (1). DOT1L is required for the initiation and maintenance of mixed lineage leukemia (MLL)-rearranged leukemias, and selective DOT1L inhibitors such as EP2-5676 show remarkable anti-tumor effects in MLL-rearranged leukemias (2,3). Multiple studies have also implicated DOT1L in solid tumor cancers such as breast cancer, where DOT1L induces neoplastic transformation of immortalized breast cancer cells and promotes tumor initiation and growth. Overexpression of DOT1L is associated with poor prognosis in breast cancer, and selective DOT1L inhibitors are able to suppress proliferation and migration of breast cancer cells (4-6).

  1. Kim, W. et al. (2012) J Biol Chem 287, 5588-99.
  2. Okuda, H. et al. (2017) J Clin Invest 127, 1918-1931.
  3. McLean, C.M. et al. (2014) Leukemia 28, 2131-8.
  4. Lee, J.Y. and Kong, G. (2015) Oncotarget 6, 30451-2.
  5. Zhang, L. et al. (2014) Oncotarget 5, 10665-77.
  6. Cho, M.H. et al. (2015) Nat Commun 6, 7821.

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 nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation ChIP: Chromatin IP 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.
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.

使用に関する制限

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Revision 5
#77087

DOT1L (D1W4Z) Rabbit mAb

Western Blotting Image 1: DOT1L (D1W4Z) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using DOT1L (D1W4Z) Rabbit mAb.
Western Blotting Image 2: DOT1L (D1W4Z) Rabbit mAb Expand Image
Western blot analysis of extracts from HCT116 cells, either untransfected (lane 1), mock transfected (lane 2), or transfected with DOT1L siRNA (lane 3) using DOT1L (D1W4Z) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
No image available
Chromatin Immunoprecipitation Image 1: DOT1L (D1W4Z) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293T cells and either DOT1L (D1W4Z) 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 SimpleChIP® Human PabPC1 Exon 1 Primers #77410, Human RPLP0 Exon 2 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.
CUT and RUN Image 1: DOT1L (D1W4Z) Rabbit mAb Expand Image
CUT&RUN was performed with 293T cells and DOT1L (D1W4Z) 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 PabPC1, a known target gene of DOT1L (see additional figure containing qPCR data).
CUT and RUN Image 2: DOT1L (D1W4Z) Rabbit mAb Expand Image
CUT&RUN was performed with 293T cells and DOT1L (D1W4Z) 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 figures show binding across chromosome 8 (upper), including PabPC1 (lower), a known target gene of DOT1L (see additional figure containing qPCR data).
CUT and RUN Image 3: DOT1L (D1W4Z) Rabbit mAb Expand Image
CUT&RUN was performed with 293T cells and either DOT1L (D1W4Z) 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 PabPC1 Exon 1 Primers #77410, SimpleChIP® Human RPL30 Exon 3 Primers #7014, 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.