Western blot shows lysates of HEK293 human embryonic kidney cell line transfected with C-terminal DYKDDDDK-tagged Abro1, N-terminal DYKDDDDK-tagged UCHL5, and N-terminal DYKDDDDK-tagged RNF20. PVDF membrane was probed with 0.1 µg/mL of Rabbit Anti-DYKDDDDK Epitope Tag Monoclonal Antibody (Catalog # MAB8529) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (HAF008). Specific bands were detected for DYKDDDDK-tagged proteins at approximately 55 kDa, 40 kDa, 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of DYKDDDDK (FLAG^®) Epitope Tag by Simple Western^TM.

Simple Western lane view shows lysates of HEK293 human embryonic kidney cell line transfected with C-terminal DYKDDDDK-tagged Abro1 and N-terminal DYKDDDDK-tagged RNF20, loaded at 0.1 mg/mL. Specific band were detected for DYKDDDDK-tagged proteins at approximately 64 and 136 kDa (as indicated) using 1 µg/mL of Rabbit Anti-DYKDDDDK Epitope Tag Monoclonal Antibody (Catalog # MAB8529). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of DYKDDDDK Epitope Tag in HEK293 cells transfected with FLAG vs Irrelevant HEK293 wild type cells by Flow Cytometry.

HEK293 cells transfected with DYKDDDDK-tagged proteins (filled histogram) vs Irrelevant HEK293 wild type cells (open histogram) were stained with Rabbit Anti-DYKDDDDK Epitope Tag Monoclonal Antibody (Catalog # MAB8529) at 0.001 ug/10⁶ cells followed by Allophycocyanin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0111). To facilitate intracellular staining, cells were fixed and permeabilized with Flow Cytometry Fixation Buffer (Catalog # FC004). View our protocol for Staining Intracellular Molecules.

Applications for DYKDDDDK Epitope Tag Antibody

Application

Recommended Usage

Immunoprecipitation

1-4 µg/100 µg cell lysate
Sample: HEK293 human embryonic kidney cell line transfected with C-terminal DYKDDDDK-tagged Abro1 and N-terminal DYKDDDDK-tagged RNF20, see our available Western blot detection antibodies.

Intracellular Staining by Flow Cytometry

0.001 µg/10⁶ cells
Sample: HEK293 cells transfected with FLAG, fixed and permeabilized with Flow Cytometry Fixation Buffer (Catalog # FC004).

Simple Western

1 µg/mL
Sample: HEK293 human embryonic kidney cell line transfected with C-terminal DYKDDDDK-tagged Abro1 and N-terminal DYKDDDDK-tagged RNF20

Western Blot

0.1 µg/mL
Sample: HEK293 human embryonic kidney cell line transfected with C-terminal DYKDDDDK-tagged Abro1, N-terminal DYKDDDDK-tagged UCHL5, and N-terminal DYKDDDDK-tagged RNF20

Reviewed Applications

Read 4 reviews rated 4 using MAB8529 in the following applications:

Immunocytochemistry (2 Reviews)
Simple Western (1 Review)
Western Blot (1 Review)

Formulation, Preparation, and Storage

Purification

Protein A or G purified from cell culture supernatant

Reconstitution

Reconstitute at 0.5 mg/mL in sterile PBS

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose.

Shipping

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

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.

12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.

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 Specific Notices for DYKDDDDK Epitope Tag Antibody

For research use only

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Flow Cytometry

DYKDDDDK Epitope Tag Antibody

R&D Systems, part of Bio-Techne | Catalog # MAB8529

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Scientific Data Images for DYKDDDDK Epitope Tag Antibody

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

Detection of DYKDDDDK (FLAG®) Epitope Tag by Simple WesternTM.