Revision 7
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, IHC-P, IF-IC, FC-FP, ChIP

REACTIVITY:

All

SENSITIVITY:

Transfected Only

MW (kDa):

Source/Isotype:

Rabbit IgG

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
Immunoprecipitation 1:50
Immunohistochemistry (Paraffin) 1:400 - 1:1600
Immunofluorescence (Immunocytochemistry) 1:400 - 1:1600
Flow Cytometry (Fixed/Permeabilized) 1:800 - 1:3200
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 –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #70586.

Specificity / Sensitivity

DYKDDDDK Tag (D6W5B) Rabbit mAb detects exogenously expressed DYKDDDDK proteins in cells. The antibody recognizes the DYKDDDDK peptide, which is the same epitope recognized by Sigma-Aldrich Anti-FLAG M2 antibody, fused to either the amino-terminus or carboxy-terminus of the target protein.

Species Reactivity:

All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic DYKDDDDK peptide.

Background

Epitope tags are useful for the labeling and detection of proteins using immunoblotting, immunoprecipitation, and immunostaining techniques. Because of their small size, they are unlikely to affect the tagged protein’s biochemical properties.

The DYKDDDDK peptide has been used extensively as a general epitope tag in expression vectors. This peptide can be expressed and detected with the protein of interest as an amino-terminal or carboxy-terminal fusion (1).

  1. Brizzard, B.L. et al. (1994) Biotechniques 16, 730-5.

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 IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) 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.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
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
#14793

DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma-Aldrich Anti-FLAG M2 antibody)

Western Blotting Image 1: DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma-Aldrich Anti-FLAG M2 antibody) Expand Image
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with DYKDDDDK-GFP (N-terminal DDK-Tag; +), GFP-DYKDDDDK (C-terminal DDK-Tag; +), human CASQ1-DYKDDDDK (C-terminal DDK-Tag; +), or human FoxG1-DYKDDDDK (C-terminal DDK-Tag; +) as indicated, using DYKDDDDK Tag (D6W5B) Rabbit mAb.
Western Blotting Image 2: DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma-Aldrich Anti-FLAG M2 antibody) Expand Image
Western blot analysis of recombinant human SYN3-MYC/DYKDDDDK at various concentrations using, DYKDDDDK Tag (D6W5B) Rabbit mAb.
Immunoprecipitation Image 1: DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma-Aldrich Anti-FLAG M2 antibody) Expand Image
Immunoprecipitation of FoxG1-DYKDDDDK tag protein from 293T transfected cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or DYKDDDDK (D6W5B) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using DYKDDDDK (D6W5B) Rabbit mAb.
Immunohistochemistry Image 1: DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma-Aldrich Anti-FLAG M2 antibody) Expand Image
Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, control (left) or DYKDDDDK Tag-transfected (right), using DYKDDDDK Tag (D6W5B) Rabbit mAb.
Immunofluorescence Image 1: DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma-Aldrich Anti-FLAG M2 antibody) Expand Image
Confocal immunofluorescent analysis of 293T cells transfected with a GFP-DYKDDDDK-Tag (green) using DYKDDDDK Tag (D6W5B) Rabbit mAb (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow Cytometry Image 1: DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma-Aldrich Anti-FLAG M2 antibody) Expand Image
Flow cytometric analysis of 293 cells, untransfected (blue) or transfected with DYKDDDDK-Tag (green), using DYKDDDDK Tag (D6W5B) Rabbit mAb. Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma-Aldrich Anti-FLAG M2 antibody) Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293T cells, untransfected (left) or transfected (right) with a DYKDDDDK-tagged SSRP1 construct and DYKDDDDK (D6W5B) Rabbit mAb, SSRP1 (E1Y8D) Rabbit mAb #13421, 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 c-Fos Exon 3 Primers #12010 and SimpleChIP® Human EGR1 Intron 3 Primers #11953. 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.