DYKDDDDK Epitope Tag Antibody (29E4.G7) [FITC]

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Product Details

Summary
Reactivity NASpecies Glossary
Applications ICC/IF
Clone
29E4.G7
Clonality
Monoclonal
Host
Mouse
Conjugate
FITC
Concentration
LYOPH

Order Details

DYKDDDDK Epitope Tag Antibody (29E4.G7) [FITC] Summary

Immunogen
This antibody was produced in mice by repeated immunizations with a synthetic peptide corresponding to the FLAG epitope tag peptide DYKDDDDK (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) conjugated to KLH using maleimide. Residues of glycine and cysteine were added to the termini to facilitate coupling.
Specificity
The purified antibody is directed against the motif and is useful in determining its presence in various assays where the epitope tag is present at either the amino or carboxy terminus of recombinant proteins. This monoclonal tag antibody detects over-expressed proteins containing the epitope tag. In Western blotting of bacterial extracts, the antibody does not cross-react with endogenous proteins.
Isotype
IgG2a Kappa
Clonality
Monoclonal
Host
Mouse
Purity
Protein A purified
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Applications/Dilutions

Dilutions
  • Immunocytochemistry/Immunofluorescence 1:500-1:2500
Application Notes
This antibody is optimally suited for monitoring the expression of FLAG tagged fusion proteins. As such, this antibody can be used - identify fusion proteins containing the FLAG epitope. The antibody recognizes the epitope tag fused - either the amino- or carboxy- termini of targeted proteins. The epitope tag peptide sequence was first derived from the 11-amino-acid leader peptide of the gene-10 product from bacteriophage T7. DYKDDDDK is the most commonly used hydrophilic octapeptide tag.

Packaging, Storage & Formulations

Storage
Store lyophilized antibody at 4C. Aliquot reconstituted liquid and store at -20C. Avoid freeze-thaw cycles.
Buffer
Lyophilized from 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2, 10 mg/mL Bovine Serum Albumin (BSA) - Immunoglobulin and Protease free
Preservative
0.01% Sodium Azide
Concentration
LYOPH
Purity
Protein A purified
Reconstitution Instructions
Reconstitute with 100 ul deionized water.

Notes

Stabilizer: 10 mg/ml Bovine Serum Albumin (BSA) - Immunoglobulin and Protease free
Color: Green Excitation Wavelength: 495 Emission Wavelength: 528
Conjugation Chemistry: Isothiocyanate

Alternate Names for DYKDDDDK Epitope Tag Antibody (29E4.G7) [FITC]

  • DDDDK epitope tag
  • DDK
  • DYKDDDDK Epitope Tag
  • DYKDDDDK tag
  • ECS epitope tag
  • ECS tag
  • Enterokinase Cleavage Site epitope tag
  • Enterokinase Cleavage Site tag
  • epitope tag flag
  • FLAG (FLAG is a trademark of Sigma Aldrich)
  • FLAG Tag
  • Ty 1 Tag
  • xxxDDDDK epitope tag

Background

Fusion proteins, in which a known peptide such as a His-Tag, GST-Tag, or DYKDDDDK (FLAG(R)) is cloned with the target of interest, were developed to simplify protein isolation. Since specific antibodies may not be available against the protein of interest, epitope tag antibodies are commonly used to detect protein expression.

The epitope tag, DYKDDDDK (FLAG(R)), is comprised of an eight amino acid sequence derived from the gene-10 product from bacteriophage T7 and contains an enterokinase-cleavage site (1). Its hydrophilic nature and short peptide sequence reduce possible interference with protein expression, post translational modifications, protein interactions, and protein function (2). The DYKDDDDK (FLAG(R)) epitope tag can either be fused to the N-terminus, C-terminus, or placed at an internal position of the target protein, facilitating characterization of the protein of interest using immunodetection methods. Compared to the standard FLAG(R) epitope tag antibody, the rat monoclonal DYKDDDDK Epitope Tag antibody (clone L5) has been shown to be 2-8 times more sensitive (3).

References

1. Brizzard BL, Chubet RG, Vizard DL. (1994) Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. Biotechniques. 16(4):730-5. PMID: 8024796

2. Einhauer A, Jungbauer A. (2001) The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Methods. 49(1-3):455-65. PMID: 11694294

3. Park SH, Cheong C, Idoyaga J, Kim JY, Choi JH, Do Y, Lee H, Jo JH, Oh YS, Im W, Steinman RM, Park CG. (2008) Generation and application of new rat monoclonal antibodies against synthetic FLAG and OLLAS tags for improved immunodetection. J Immunol Methods. 331(1-2):27-38. PMID: 18054954

FLAG(R) is a trademark of Sigma-Aldrich

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Product General Protocols

Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

Video Protocols

ICC/IF Video Protocol

FAQs for DYKDDDDK Epitope Tag Antibody (NBP1-97401). (Showing 1 - 8 of 8 FAQ).

  1. Are the FLAG Tag antibodies validated in Simple Western?
    • Yes, we offer a 2 FLAG Tag antibodies that have been tested in Simple Western: NB600-345 and NBP1-71705.
  2. Does FLAG Tag antibodies comes in lyophilized form?
    • The following FLAG Tag antibodies come in lyophilized form: NBP1-97401, NBP1-97399, NBP1-97400, NBP1-97402.
  3. What the theoretical molecular weight for FLAG Tag antibodies?
    • The TMW of FLAG Tag antibodies is approximately 150 kDa.
  4. Will FLAG Tag antibodies detect internal FLAG tags? My FLAG sequence is sandwiched between two other protein sequences.
    • If you are applying your sample(s) to SDS-PAGE, under reducing condition and follow by Western Blot, DYKDDDDK epitope tag antibodies should be able to detect the tag region located internally. However, it depends on the property of your chimeric protein and if your tag is in the hydrophobic or hydrophilic region. Although DYKDDDDK is very hydrophilic, it may not be exposed to the surface if embedded deeply inside the protein with a high content of hydrophobic amino acids with or without covalently-linked modifications such as lipid moieties.
  5. Will FLAG Tag antibodies recognizes FLAG at the C-terminal end?
    • Yes, FLAG Tag antibodies can recognize FLAG-tag at both the N- or C-terminal.
  6. Hello. We are planning to promote DYKDDDDK Antibodies in our February 2013 newsletter. Thus, we would like to check toxicity of a few products. Could you let us know whether these are lyophilized form?
    • NBP1-97401 does not come in lyophilized form.
  7. Does this FLAG Tag antibody come in lyophilized form?
    • Yes, we carry this FLAG Tag antibody in lyophilized format.
  8. What is the immunogen sequence of this FLAG Tag antibody?
    • A synthetic peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) corresponding to the FLAG epitope tag peptide DYKDDDDK, conjugated to KLH.

Secondary Antibodies

 

Isotype Controls

Other Available Formats

Alexa Fluor 350 NBP1-97410AF350
Alexa Fluor 405 NBP1-97410AF405
Alexa Fluor 488 NBP1-97410AF488
Alexa Fluor 532 NBP1-97410AF532
Alexa Fluor 594 NBP1-97410AF594
Alexa Fluor 647 NBP1-97410AF647
Alexa Fluor 700 NBP1-97410AF700
Alexa Fluor 750 NBP1-97410AF750
Allophycocyanin NBP1-97410APC
Biotin NBP1-97400
Biotin NBP1-97410B
DyLight 350 NBP1-97410UV
DyLight 405 NBP1-97399
DyLight 405 NBP1-97410V
DyLight 488 NBP1-97410G
DyLight 550 NBP1-97410R
DyLight 594 NBP1-97410DL594
DyLight 650 NBP1-97410C
DyLight 680 NBP1-97410FR
DyLight 755 NBP1-97410IR
FITC NBP1-97410F
FITC NBP1-97401
HRP NBP1-97410H
HRP NBP1-97393
Janelia Fluor 549 NBP1-97410JF549
Janelia Fluor 646 NBP1-97410JF646
PE NBP1-97410PE
PerCP NBP1-97410PCP

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