Western blot shows lysates of HEK293 human embryonic kidney cell line transfected with C-terminal DYKDDDDK-tagged Abro1 and N-terminal DYKDDDDK-tagged RNF20. PVDF membrane was probed with 1:1000-1:3000 dilution of Rabbit Anti-DYKDDDDK Epitope Tag HRP-conjugated Monoclonal Antibody (Catalog # HAM85291). Specific bands were detected for DYKDDDDK-tagged proteins at approximately 55 kDa and 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Applications for DYKDDDDK Epitope Tag Horseradish Peroxidase-conjugated Antibody

Application

Recommended Usage

Western Blot

1:1000-1:3000 dilution
Sample: HEK293 human embryonic kidney cell line transfected with C-terminal DYKDDDDK-tagged Abro1 and N-terminal DYKDDDDK-tagged RNF20

Formulation, Preparation, and Storage

Purification

Protein A or G purified from cell culture supernatant

Formulation

Supplied as a 0.2 μm filtered solution in PBS

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Protect from light. Do not freeze.

12 months from date of receipt, 2 to 8 °C as supplied.

Background: DYKDDDDK Epitope Tag

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, 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 DYKDDDDK epitope tag antibody, the rat monoclonal DYKDDDDK Epitope Tag antibody (clone L5) is 2-8 times more sensitive (3).

References

1. Brizzard BL, Chubet RG, Vizard DL. (1994) Immunoaffinity purification of (FLAG(R)) 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(R)) 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 DYKDDDDK Epitope Tag (FLAG(R)) and OLLAS tags for improved immunodetection. J Immunol Methods. 331(1-2):27-38. PMID: 18054954

FLAG(R) is a trademark of Sigma-Aldrich.

Alternate Names

FLAG Tag

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Product Documents for DYKDDDDK Epitope Tag Horseradish Peroxidase-conjugated Antibody

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Product Specific Notices for DYKDDDDK Epitope Tag Horseradish Peroxidase-conjugated Antibody

FLAG is a registered trademark of SigmaAldrich Co. LLC

For research use only

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DYKDDDDK Epitope Tag Horseradish Peroxidase-conjugated Antibody

R&D Systems, part of Bio-Techne | Catalog # HAM85291

Key Product Details

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Product Specifications

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Specificity

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Scientific Data Images for DYKDDDDK Epitope Tag Horseradish Peroxidase-conjugated Antibody

Detection of DYKDDDDK (FLAG®) Epitope Tag by Western Blot.