Invitrogen

DYKDDDDK Tag Recombinant Polyclonal Antibody (20H18L16, 20H1L23, 8H2L5, 8H8L17)

Advanced Verification

This Antibody was verified by Relative expression to ensure that the antibody binds to the antigen stated.

2 Published Figures

8 References

View all (104) DYKDDDDK Tag antibodies

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Datasheet

Protocols

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DYKDDDDK Tag Antibody in Immunocytochemistry (ICC/IF) — For immunofluorescence analysis DYKDDDDK - BNIP3 transfected HEK293 cells were fixed and permeabilized for detection of DYKDDDDK-Tag protein using Anti-DYKDDDDK Recombinant Rabbit Polyclonal Antibody (Product # 740001, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of DYKDDDDK-Tag protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of DYKDDDDK - BNIP3. Panel e) shows no signal with untransfected cell and panel f) represents control cells with no primary antibody to assess background.

DYKDDDDK Tag Antibody in Western Blot (WB) — Western blot was performed using Anti-DYKDDDDK Tag Recombinant Polyclonal Antibody (20H18L16, 20H1L23, 8H2L5, 8H8L17) (Product # 740001) by loading whole cell extracts of untransfected and transiently transfected HEK-293E lysates: untransfected, 50 µg (Lane 1), empty vector control, 50 µg (Lane 2), HA-H3-FLAG, 40 µg (Lane 3), HA-H3-FLAG, 20 µg (Lane 4), HA-H3-FLAG, 10 µg (Lane 5), HA-H3-FLAG, 5 µg (Lane 6), FLAG-p65-HA, 40 µg (Lane 7), Myc-p65-V5, 40 µg (Lane 8) and 25 ng of Positope (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). A ~17 kDa band corresponding to HA-H3-FLAG and ~67 kDa band corresponding to FLAG-p65-HA were observed in HEK293E transfected lysates on probing with the primary antibody (1 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1500 (Product # A44241). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Positope (Product # R90050) is a 53 kDa recombinant protein consisting multiple epitope tags but lacks FLAG tag, which has been used as a negative control for FLAG tag detection. No cross-reactivity was

DYKDDDDK Tag Antibody in Immunoprecipitation (IP) — Immunoprecipitation of DYKDDDDK tagged BNIP3 was performed with 1 µg/mL of the Anti-DYKDDDDK Recombinant Rabbit Polyclonal Antibody (Product # 740001) on cell extract from DYKDDDDK-BNIP3 transfected HEK-293 cells using the Dynabeads® Protein A Immunoprecipitation Kit (Product # 10006D). Normal Rabbit IgG was used as a negative IP control. Subsequently, Western blot analysis was performed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0341BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Proteins were transferred using iBlot® 2 Dry Blotting System (Product # IB21001) to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature on a rocking platform. DYKDDDDK-BNIP3 was detected at ~35 kDa as homodimer and ~18 kDA as monomer using DYKDDDDK Recombinant Rabbit Polyclonal Antibody (Product # 740001) at 1 µg/mL in 5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution) was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106). Lane 1 represents 10% of the total cell extract (input), Lane 2 is the IP performed with Rabbit IgG and Lane 3 represents IP performed with Anti-DYKDDDDK Recombinant Rabbit Polyclonal Antibody.

DYKDDDDK Tag Antibody — Antibody specificity was demonstrated by detection of different targets fused to DYKDDDDK tag in transiently transfected lysates tested. Relative detection of DYKDDDDK tag was observed across different proteins fused with DYKDDDDK tag in HA-H3-DYKDDDDK (Lane 3-6) and DYKDDDDK-p65-HA (Lane 7), using Anti-DYKDDDDK Tag Recombinant Polyclonal Antibody (20H18L16, 20H1L23, 8H2L5, 8H8L17) (Product # 740001) in Western Blot. This product has been shown to detect DYKDDDDK Tag at both N- and C- termini of a fusion protein. {RE}

Product Details

740001

Applications	Tested Dilution	Publications
Western Blot (WB)	0.5-1 µg/mL	View 3 publications 3 publications
Immunocytochemistry (ICC/IF)	1 µg/mL	View 5 publications 5 publications
Immunoprecipitation (IP)	1 µg/mL	-

Product Specifications
Species Reactivity	Tag
Published species	Tag
Host/Isotype	Rabbit / IgG
Class	Recombinant Polyclonal
Type	Antibody
Clone	20H18L16, 20H1L23, 8H2L5, 8H8L17
Immunogen	Synthetic peptide (DYKDDDDK)
Conjugate	Unconjugated
Form	Liquid
Concentration	0.5 mg/mL
Purification	Protein A
Storage buffer	PBS, pH 7.2
Contains	0.09% sodium azide
Storage conditions	Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
RRID	AB_2610628

Product Specific Information

This antibody consists of four clones viz. 20H18L16 and 20H1L23 from the N terminal and 8H2L5 and 8H8L17 from the C terminal.

Recombinant rabbit polyclonal antibodies are unique offerings from Thermo Fisher Scientific. They are comprised of a mixture of recombinant antibodies co-expressed from a library of heavy and light chains, providing the best of both worlds – the sensitivity of polyclonal antibodies with the specificity of monoclonal antibodies - all delivered with the consistency only found in a recombinant antibody. While functionally the same as a polyclonal antibody – recognizing multiple epitope sites on the target and producing higher detection sensitivity for low abundance targets – the exact population of the heavy and light chains in a recombinant rabbit polyclonal antibody can be produced in every lot, circumventing the biological variability typically associated with polyclonal antibody production.

Target Information

DYKDDDDK Tag (FLAG® tag, FLAG octapeptide) is a polypeptide protein tag that can be added to a protein using recombinant DNA technology. The FLAG tag structure has been optimized for compatibility with the proteins it is attached to, in that it is more hydrophilic than other common epitope tags and therefore less likely to denature or inactivate proteins to which it is appended. A FLAG tag can be used in many different assays that require recognition by an antibody. If there is no antibody against the studied protein, adding a FLAG tag to this protein allows one to follow the protein with an antibody against the FLAG sequence. Examples are cellular localization studies by immunofluorescence or detection by SDS PAGE protein electrophoresis. Tagging with xxxDDDDK may be done at the N-terminus, N-terminus preceded by a methionine residue, C-terminus, and in internal positions of the target protein. The small size of the epitope tag and its high hydrophilicity tend to decrease the possibility of interference with protein expression, proteolytic maturation, antigenicity and function. The enterokinase cleavage site allows it to be completely removed from the purified fusion proteins.

FLAG is a registered trademark of Merck KGaA, Darmstadt, Germany.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Product References

Manipulation of Promyelocytic Leukemia Protein Nuclear Bodies by Marek's Disease Virus Encoded US3 Protein Kinase.

Microorganisms

Liao Y,Lupiani B,Reddy SM

Published figure using DYKDDDDK Tag recombinant polyclonal antibody (Product # 740001) in Immunocytochemistry

Fri Mar 26 00:00:00 EDT 2021

Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics.

Cell

Linghu C,Johnson SL,Valdes PA,Shemesh OA,Park WM,Park D,Piatkevich KD,Wassie AT,Liu Y,An B,Barnes SA,Celiker OT,Yao CC,Yu CJ,Wang R,Adamala KP,Bear MF,Keating AE,Boyden ES

740001 was used in Immunocytochemistry to create SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus.

Thu Dec 10 00:00:00 EST 2020

An interaction of heart disease-associated proteins POPDC1/2 with XIRP1 in transverse tubules and intercalated discs.

BMC molecular and cell biology

Holt I,Fuller HR,Schindler RFR,Shirran SL,Brand T,Morris GE

740001 was used in Immunocytochemistry-immunoflourescence to identify novel protein interaction partners of Popeye domain-containing proteins 1 and 2.

Tue Dec 01 00:00:00 EST 2020

The Vaccinia Virus (VACV) B1 and Cellular VRK2 Kinases Promote VACV Replication Factory Formation through Phosphorylation-Dependent Inhibition of VACV B12.

Journal of virology

Rico AB,Wang Z,Olson AT,Linville AC,Bullard BL,Weaver EA,Jones C,Wiebe MS

740001 was used in Immunocytochemistry-immunoflourescence to provide evidence of how poxviruses can be multifaceted in their mimicry of cellular proteins through the consolidation of functions of both VRK1 and VRK2 within the viral B1 protein kinase.

Tue Oct 15 00:00:00 EDT 2019

Identification of ARKL1 as a Negative Regulator of Epstein-Barr Virus Reactivation.

Journal of virology

Siddiqi UZ,Vaidya AS,Li X,Marcon E,Tsao SW,Greenblatt J,Frappier L

740001 was used in Immunocytochemistry-immunoflourescence to identify the cellular Arkadia-like 1 (ARKL1) protein as a negative regulator of Zp and EBV reactivation.

Tue Oct 15 00:00:00 EDT 2019

Identification of ARKL1 as a Negative Regulator of Epstein-Barr Virus Reactivation.

Journal of virology

Siddiqi UZ,Vaidya AS,Li X,Marcon E,Tsao SW,Greenblatt J,Frappier L

740001 was used in Immunocytochemistry-immunoflourescence to identify the cellular Arkadia-like 1 (ARKL1) protein as a negative regulator of Zp and EBV reactivation.

Tue Oct 15 00:00:00 EDT 2019

HDAC2 Regulates Site-Specific Acetylation of MDM2 and Its Ubiquitination Signaling in Tumor Suppression.

iScience

Patel N,Wang J,Shiozawa K,Jones KB,Zhang Y,Prokop JW,Davenport GG,Nihira NT,Hao Z,Wong D,Brandsmeier L,Meadows SK,Sampaio AV,Werff RV,Endo M,Capecchi MR,McNagny KM,Mak TW,Nielsen TO,Underhill TM,Myers RM,Kondo T,Su L

740001 was used in Western Blotting to uncover a new HDAC2-dependent pathway that integrates reversible acetylation signaling to the anticancer ubiquitin response.

Fri Mar 29 00:00:00 EDT 2019

An efficient and quantitative assay for epitope-tagged therapeutic protein development with a capillary western system.

Bioanalysis

Li Y,Duah E,Long N,Persaud A,VanGundy Z,Magliery T,Poi MJ

740001 was used in Simple Western - Size to develop and validate a reliable, robust and efficient assay to detect and quantify biologic compounds in vitro and in vivo during early stage of a biotherapeutic agent discovery.

Fri Mar 01 00:00:00 EST 2019

Bioinformatics

Protein Aliases: DDDDK; DDDDK epitope tag; ECS epitope tag; ECS tag; FLAG; FLAG Tag

DYKDDDDK Tag Recombinant Polyclonal Antibody (20H18L16, 20H1L23, 8H2L5, 8H8L17)

Product Details

740001

Western Blot (WB)

Immunocytochemistry (ICC/IF)

Immunoprecipitation (IP)

Species Reactivity

Published species

Host/Isotype

Class

Type

Clone

Immunogen

Conjugate

Form

Concentration

Purification

Storage buffer

Contains

Storage conditions

RRID

Product Specific Information

Target Information

Bioinformatics

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