DYKDDDDK Tag (FLAG) Mouse Monoclonal Antibody [A2-A4]

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1ml

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Catalog# M1403-2

DYKDDDDK Tag (FLAG) Mouse Monoclonal Antibody [A2-A4]

Application

WB
IP
ELISA
IF-Cell

Reactivity

Species independent

This product has been cited in peer reviewed publications, see list HERE

概述

产品名称

DYKDDDDK Tag (FLAG) Mouse Monoclonal Antibody [A2-A4]

抗体类型

Mouse Monoclonal Antibody

免疫原

Synthetic peptide immune sequence is N-DYKDDDDK-C.

种属反应性

Species independent

验证应用

WB, IP, ELISA, IF-Cell

阳性对照

DYKDDDDK Tag recombinant protein, C-terminal FLAG-tagged recombinant protein, N-terminal FLAG-tagged recombinant protein.

偶联

unconjugated

克隆号

A2-A4

RRID

AB_3073075

产品特性

形态

Liquid

浓度

2ug/ul

存放说明

Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.

存储缓冲液

1*PBS (pH7.4), 0.2% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

亚型

IgG1

纯化方式

Protein G affinity purified.

应用稀释度

WB
1:5,000-1:10,000

IP
2-5 µg/ml.

IF-Cell
1:100

发表文章中的应用

WB	See 38 publications below
Co-IP	See 11 publications below
IP	See 6 publications below

发表文章中的种属

Human	See 26 publications below
Mouse	See 8 publications below
HeLa cells	See 2 publications below

靶点

功能

FLAG-tag, or FLAG octapeptide, or FLAG epitope, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology, having the sequence motif DYKDDDDK (where D=aspartic acid, Y=tyrosine, and K=lysine). It is one of the most specific tags and it is an artificial antigen to which specific, high affinity monoclonal antibodies have been developed and hence can be used for protein purification by affinity chromatography and also can be used for locating proteins within living cells. It has been used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. It can also be used in the isolation of protein complexes with multiple subunits, because its mild purification procedure tends not to disrupt such complexes. It has been used to obtain proteins of sufficient purity and quality to carry out 3D structure determination by x-ray crystallography. A FLAG-tag can be used in many different assays that require recognition by an antibody. If there is no antibody against a given protein, adding a FLAG-tag to a protein allows the protein to be studied with an antibody against the FLAG sequence. Examples are cellular localization studies by immunofluorescence, immunoprecipitation or detection by SDS PAGE protein electrophoresis and Western blotting. The peptide sequence of the FLAG-tag from the N-terminus to the C-terminus is: DYKDDDDK (1012 Da). Additionally, it may be used in tandem, commonly the 3xFLAG peptide: DYKDHD-G-DYKDHD-I-DYKDDDDK (with the final tag encoding an enterokinase cleavage site). It can be fused to the C-terminus or the N-terminus of a protein, or inserted within a protein. The tyrosine residue in the FLAG-tag can be sulfated, which can affect antibody recognition of the FLAG epitope. The FLAG-tag can be used in conjunction with other affinity tags, for example a polyhistidine tag (His-tag), HA-tag or myc-tag.

背景文献

暂无

别名

DDDDK epitope tag antibody

DDDK antibody

ddk antibody

DYKDDDDK antibody

DYKDDDDK epitope tag antibody

DYKDDDDK tag antibody

ECS epitope tag antibody

ECS tag antibody

Enterokinase Cleavage Site epitope tag antibody

Enterokinase Cleavage Site tag antibody

展开

图片

Western blot analysis of DYKDDDDK Tag on Flag-tagged recombinant protein Flag-Ki67 and Ki67-Flag at different cell lysates level.

Predicted band size: 35 kDa;
Observed band size: 35 kDa;
Exposure time: 60 seconds;
SDS-PAGE gel concentration:12%

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (M1403-2, 1/2,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:200,000 dilution was used for 1 hour at room temperature.
Western blot analysis of DYKDDDDK Tag on Flag-tagged recombinant protein 2xflag-Histone H3.1 at different dilutions of HeLa cells with anti-DYDDDDK antibody (M1403-2).

Lane 1: Non-transfected HeLa cell lysates (1/2000)
Lane 2~4: Transfected HeLa cell lysates (1/2500; 1/5000; 1/10,000)
Predicted band size: 15 kDa;
Observed band size: 18 kDa;
Exposure time: 30 seconds;
SDS-PAGE gel concentration:12%.

Cell lysate at 10ug per lane.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (M1403-2) was used at different dilutions in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:200,000 dilution was used for 1 hour at room temperature.
Immunoprecipitation analysis of N-Flag in HeLa cells.

Lane 1: HeLa transfected with DYKDDDDK-tagged human Histone H3.1 expression vector whole cell lysate (input).
Lane 2: M1403-2 IP in HeLa transfected with DDDDK-tagged human Histone H3.1 expression vector whole cell lysate.
Lane 3: Mouse IgG (HA1027) instead of M1403-2 in HeLa transfected with DYKDDDDK-tagged human Histone H3.1 expression vector whole cell lysate.

Predicted band size: 19 kDa;
Observed band size: 19 kDa;
Exposure time: 60 seconds;

SDS-PAGE gel concerntration: 15%.

DYKDDDDK Tag was immunoprecipitated from 0.5 mg Hela transfected with human 2x Flag- Histone H3.1 expression vector whole cell lysate with M1403-2 2 ug/mL. Western blot was performed from the immunoprecipitate using M1403-2 at 1/5,000 dilution for 1 hour at room temperature. The secondary antibody for IP detection reagent, Goat anti-Mouse IgG-HRP antibody (HA1006), was used at 1:100,000 dilution.
Immunoprecipitation analysis of C-Flag in HeLa cells.

Lane 1: HeLa transfected with DYKDDDDK-tagged human Siglec15 expression vector whole cell lysate (input).
Lane 2: M1403-2 IP in HeLa transfected with DDDDK-tagged human Siglec15 expression vector whole cell lysate.
Lane 3: Mouse IgG (HA1027) instead of M1403-2 in HeLa transfected with DYKDDDDK-tagged human Siglec15 expression vector whole cell lysate.

DYKDDDDK Tag was immunoprecipitated from 0.5 mg Hela transfected with human Siglec15 expression vector whole cell lysate with M1403-2 2 ug/mL. Western blot was performed from the immunoprecipitate using M1403-2 at 1/2,000 dilution for 1 hour at room temperature. The secondary antibody for IP detection reagent, Goat anti-Mouse IgG-HRP antibody (HA1006), was used at 1:100,000 dilution.
Immunofluorescent analysis of 2xFlag tagged Histone H3.1 in HeLa cells.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labeling DYKDDDDK tag with M1403-2 at 1/100 dilution, followed by iFluor™ 488 Goat anti-mouse IgG antibody (HA1125) at 1/1000 dilution (green).