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Ultra-LEAF™ Purified anti-DYKDDDDK Epitope Tag Antibody

Pricing & Availability
Clone
W22083A (See other available formats)
Regulatory Status
RUO
Other Names
FLAG, FLAG tag
Isotype
Rat IgG2b, κ
Ave. Rating
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Product Citations
publications
A. W22083A_ULEAF_Epitope-Tag_WB_102723
Whole cell extracts (1.5 µg total protein) from CHO cells transfected with or without a multi-tag construct containing the DYKDDDDK epitope, and Recombinant Posi-Tag Epitope Tag Protein (15 µg) (Cat. No. 931301) were resolved by 4-12% Bis-Tris gel electrophoresis, transferred to a PVDF membrane, and probed with 0.25 µg/mL of Purified anti-DYKDDDDK Epitope Tag (clone W22083A) overnight at 4°C. Proteins were visualized by chemiluminescence detection using HRP Goat anti-rat IgG (Cat. No. 405405) at a 1:3000 dilution. Direct-Blot™ HRP anti-β-actin (Cat. No. 643807) was used as a loading control at a 1:10000 dilution. Western-Ready™ ECL Substrate Plus Kit (Cat. No. 426316) was used as a detection agent. The recombinant DYKDDDDK-tagged multi-tag and Recombinant Posi-Tag Epitope Tag Proteins (Cat. No. 931301) have an expected molecular weight of 30 kD and 48 kD, respectively. Lane M: Molecular weight marker
  • A. W22083A_ULEAF_Epitope-Tag_WB_102723
    Whole cell extracts (1.5 µg total protein) from CHO cells transfected with or without a multi-tag construct containing the DYKDDDDK epitope, and Recombinant Posi-Tag Epitope Tag Protein (15 µg) (Cat. No. 931301) were resolved by 4-12% Bis-Tris gel electrophoresis, transferred to a PVDF membrane, and probed with 0.25 µg/mL of Purified anti-DYKDDDDK Epitope Tag (clone W22083A) overnight at 4°C. Proteins were visualized by chemiluminescence detection using HRP Goat anti-rat IgG (Cat. No. 405405) at a 1:3000 dilution. Direct-Blot™ HRP anti-β-actin (Cat. No. 643807) was used as a loading control at a 1:10000 dilution. Western-Ready™ ECL Substrate Plus Kit (Cat. No. 426316) was used as a detection agent. The recombinant DYKDDDDK-tagged multi-tag and Recombinant Posi-Tag Epitope Tag Proteins (Cat. No. 931301) have an expected molecular weight of 30 kD and 48 kD, respectively. Lane M: Molecular weight marker
  • B. W22083A_ULEAF_Epitope-Tag_ICC_102723
    CHO cells untransfected (panel A) and transfected (panel B) with a multi-tag construct containing the DYKDDDDK epitope were fixed with 4% PFA Fixation Buffer (Cat. No. 420801) for 10 minutes and permeabilized with 100% ice-cold methanol for 10 minutes, and blocked with 5% FBS for 1 hour at room temperature. Cells were then stained with 1.0 µg/mL of Purified anti-DYKDDDDK Epitope Tag (clone W22083A), followed by incubation with Alexa Fluor® 594 Goat anti-rat IgG (Cat. No. 405422) for 1 hour at room temperature. Nuclei were counterstained with DAPI (Cat. No. 422801). The images were captured on a Revvity Operetta CLS™ High Content Analysis System with a 60X objective. Scale bar: 50 µm
  • C. W22083A_ULEAF_Epitope-Tag_IP_102723
    Whole cell extracts (250 µg total protein) prepared from CHO cells transfected and untransfected with a multi-tag construct containing the DYKDDDDK epitope and treated with Brefeldin A (Cat. No. 420601) were immunoprecipitated overnight with 2.5 µg of Purified Rat IgG2b, κ Isotype Control (Cat. No. 400602) or Purified anti-DYKDDDDK Epitope Tag (clone W22083A). The resulting IP fractions and whole cell extract input (6%) were resolved by 4-12% Bis-Tris gel electrophoresis, transferred to a PVDF membrane and probed with Purified anti-DYKDDDDK Epitope Tag (clone L5). Lane M: Molecular weight marker
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624451 25 µg £89
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624452 100 µg £158
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Description

The DYKDDDDK tag, commonly referred to as Sigma®'s FLAG® Tag, is often used as a protein modification in order to simplify the labeling and detection of proteins. This unique amino acid sequence allows for specific antibody detection in western blotting and immunocytochemistry. Due to the short sequence, this modification is not likely to affect the structure or function of the modified proteins.

Product Details
Technical Data Sheet (pdf)

Product Details

Verified Reactivity
Epitope tag
Reported Reactivity
Species independent
Antibody Type
Monoclonal
Host Species
Rat
Immunogen
DYKDDDDK peptide conjugated to KLH
Formulation
0.2 µm filtered in phosphate-buffered solution, pH 7.2, containing no preservative.
Endotoxin Level
Less than 0.01 EU/µg of the protein (< 0.001 ng/µg of the protein) as determined by the LAL test.
Preparation
The Ultra-LEAF™ (Low Endotoxin, Azide-Free) antibody was purified by affinity chromatography.
Concentration
The antibody is bottled at the concentration indicated on the vial, typically between 2 mg/mL and 3 mg/mL. To obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.
Storage & Handling
The antibody solution should be stored undiluted between 2°C and 8°C. This Ultra-LEAF™ solution contains no preservative; handle under aseptic conditions.
Application

WB - Quality tested
ICC, IP - Verified
Recommended Usage

Each lot of this antibody is quality control tested by western blotting. For western blotting, the suggested use of this reagent is 0.01 - 0.25 µg/mL. For immunocytochemistry, a concentration range of 0.05 - 1.0 μg/mL is recommended. For immunoprecipitation, the suggested use of this reagent is 2.5 µg/test. It is recommended that the reagent be titrated for optimal performance for each application.

Application Notes

W22083A is not suitable for ICFC application. 

For ICC, fixation permeabilization with 100% ice-cold methanol, or with 4% PFA followed by 100% ice-cold methanol is recommended at the indicated concentration. Fixation/permeabilization with 4% PFA followed by 0.5% Triton-X results in dimmer staining, with an optimal concentration in the 0.1 - 0.5 µg/mL range.

For use of this antibody (clone W22083A) in Western blot, this antibody has been demonstrated to have approximately 25% better sensitivity than commonly used anti-DYKDDDDK clone L5.

For use of this antibody (clone W22083A) in ICC, this antibody has increased background staining compared to commonly used anti-DYKDDDDK (clone L5).

RRID
AB_3083222 (BioLegend Cat. No. 624452)

Antigen Details

Structure
Epitope tag
Biology Area
Protein Purification, Western Blot Controls
Antigen References
  1. Rogers EJ, et al. 2019. Front Mol Neurosci. 12:43.  
  2. Li Y, et al. 2008. J Biol Chem. 283:22443-22456.
Gene ID
NA
UniProt
View information about Epitope tag on UniProt.org

Related FAQs

Do you guarantee that your antibodies are totally pathogen free?

BioLegend does not test for pathogens in-house aside from the GoInVivo™ product line. However, upon request, this can be tested on a custom basis with an outside, independent laboratory.

Does BioLegend test each Ultra-LEAF™ antibody by functional assay?

No, BioLegend does not test Ultra-LEAF™ antibodies by functional assays unless otherwise indicated. Due to the possible complexities and variations of uses of biofunctional antibodies in different assays and because of the large product portfolio, BioLegend does not currently perform functional assays as a routine QC for the antibodies. However, we do provide references in which the antibodies were used for functional assays and we do perform QC to verify the specificity and quality of the antibody based on our strict specification criteria.

Does BioLegend test each Ultra-LEAF™ antibody for potential pathogens?

No, BioLegend does not test for pathogens in-house unless otherwise indicated.  However, we can recommend an outside vendor to perform this testing as needed.

Have you tested this Ultra-LEAF™ antibody for in vivo or in vitro applications?

We don't test our antibodies for in vivo or in vitro applications unless otherwise indicated. Depending on the product, the TDS may describe literature supporting usage of a particular product for bioassay. It may be best to further consult the literature to find clone specific information.

Go To Top Version: 1    Revision Date: 10/30/2023

For Research Use Only. Not for diagnostic or therapeutic use.

 

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

 

BioLegend, the BioLegend logo, and all other trademarks are property of BioLegend, Inc. or their respective owners, and all rights are reserved.

 

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Toll-Free Phone: 1-877-Bio-Legend (246-5343) Phone: (858) 768-5800 Fax: (877) 455-9587

This data display is provided for general comparisons between formats.
Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors.
If you need assistance with selecting the best format contact our expert technical support team.

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