Anti-DYKDDDDK (FLAG) tag小鼠单克隆抗体

产品名称：Anti-DYKDDDDK (FLAG) tag小鼠单克隆抗体

货号： AT0022

品牌：Engibody

简介：FLAG标签抗体，亲和力高，特异性好，适用于检测细菌、酵母、昆虫细胞或哺乳动物细胞表达的包含FLAG标签的融合蛋白，无论FLAG标签处于融合蛋白的N端，C端还是中间，都可以

一管搞定全部实验（WB，IHC，ICC，IF及IP/CoIP）

Epitope tags are useful for the labeling and detection of proteins using immunoblotting, immunoprecipitation, and immunostaining techniques. Because of their small size, they are unlikely to affect the tagged protein’s biochemical properties.

The DYKDDDDK peptide has been used extensively as a general epitope tag in expression vectors. This peptide can be expressed and detected with the protein of interest as an amino-terminal or carboxy-terminal fusion.

Description

Mouse monoclonal antibody to DYKDDDDK tag - Epitope Tag Antibody - (Binds to same epitope as Sigma's Anti-FLAG M2 Antibody)

Reactivity

All Species Expected

Tested applications

WB    : 1/2000-1/10000.

IHC    : 1/200-1/5000.

ICC/IF    : 1/200-1/2000.

IP/CoIP    : 1/100-1/500.

Not yet tested in other applications. Optimal dilutions/concentrations should be determined by the end user.

Specificity

This antibody detects exogenously expressed DYKDDDDK proteins in cells. The antibody recognizes the DYKDDDDK peptide, which is the same epitope recognized by Sigma's Anti-FLAG® antibodies, fused to either the amino-terminus or carboxy-terminus of the target protein.

Immunogen

Synthetic peptide DYKDDDDK conjugated to KLH

Clonality

Monoclonal, clone number: 3A6

Isotype

Mouse IgG2a

Form

Liquid, 100 μg at 1mg/mL

Mouse monoclonal immunoglobulin in phosphate buffered saline (PBS), pH 7.3 (+/- 0.1), 50% glycerol with 0.1mg/mL BSA (IgG, protease free) as a stabilizer and 0.02% sodium azide as preservative.

Storage instruction

Store at +4°C short term (1-2 weeks). Aliquot and store at -20°C, Avoid freeze / thaw cycle.

Database links

Sequence: DYKDDDDK

Host

Mouse

Troubleshooting Guide

Western Blot Troubleshooting

1. No signal

2. High background

3. Non specific bands

4. Incorrect band size

5. White bands on black/grey blot

6. ‘Smear’

1. Troubleshooting:  NO SIGNAL

Sample preparation

• Untested species? Positive control. Check alignment.

• Not enough antigen in the sample or loaded onto the gel.

Transfer to the membrane

• Poor transfer to membrane.

• Excessive washing.

Blocking

• Too much blocking. Optimize blocking agent, concentration and time

• Cross-reaction blocking agent / antibody.

Antibody incubation and detection

• Not enough antibody.

• Incorrect secondary antibody.

• Detection kit is old - substrate inactive.

2. Troubleshooting:  HIGH BACKGROUND

Transfer to the membrane

• Choice of membrane.

Blocking

• Need to block for longer.

• Optimize blocking agent, concentration.

• Cross-reaction between blocking agent and primary or secondary.

• Phospho-specific proteins: use Casein to block.

Antibody incubation and detection

• Primary antibody concentration too high.

• Incubation temperature too high.

3. Troubleshooting:  NON SPECIFIC BANDS

Sample preparation

• Cell lines can accumulate differences in their protein expression profiles.

• Protein has multiple modified forms, altering mobility.

• Target protein is in fragments – degradation by proteases or cleaved.

• Splice variants / isoforms or possibly proteins from the same family.

• Multimers (dimers) of protein (reduce and denature).

• Bands may be non-specific. Run a no primary control

Blocking

• Insufficient blocking. Optimizing concentration, time.

Antibody incubation and detection

• Primary or secondary antibody concentration too high.

• Antibody has not been purified.

4. Troubleshooting:  INCORRECT BAND SIZE

Sample preparation

• Ensure sample is reduced and denatured unless stated otherwise on the datasheet.

• Check for isoforms.

• Is the sample a recombinant fragment? These will run at a different size.

5. Troubleshooting:  WHITE BANDS/ BLACK BLOT

• Too much primary and/or too much secondary antibody.

6. Troubleshooting: ‘Smear’

• Overloading, Protein degradation.

表位标签可用于使用免疫印迹、免疫沉淀和免疫染色技术标记和检测蛋白质。由于其体积小，它们不太可能影响标记蛋白的生化特性。

DYKDDDDK肽已被广泛用作表达载体中的通用表位标签。该肽可作为氨基末端或羧基末端融合物与感兴趣的蛋白质表达和检测。

描述

抗DYKDDDDK标签的小鼠单克隆抗体-表位标签抗体-（与Sigma的抗FLAG M2抗体结合相同的表位）

反应性

预计所有物种（转染并表达了flag标签的融合蛋白）

已测试的应用实验

WB:1/2000-1/10000。

IHC:1/200-1/5000。

ICC/IF:1/2000-1/2000。

IP/CoIP:1/100-1/500。

尚未在其他应用中测试。最佳稀释/浓度应由最终用户确定。

特异性

该抗体检测细胞中外源表达的DYKDDDDK蛋白。抗体识别DYKDDDDK肽，该肽与Sigma的Anti-FLAG®抗体识别的表位相同，融合到目标蛋白的氨基末端或羧基末端。

免疫原

与KLH偶联的合成肽DYKDDDDK

克隆性

单克隆，克隆号：3A6

同型对照

小鼠IgG2a

类型

液体，100μg，1mg/mL

小鼠单克隆免疫球蛋白，磷酸盐缓冲盐水（PBS），pH 7.3（+/-0.1），50%甘油，0.1mg/mL BSA（IgG，无蛋白酶）作为稳定剂，0.02%防腐剂。

存储说明

储存在-20°C下，避免冷冻/解冻循环。

序列：

DYKDDDDK

抗体来源宿主

小鼠

Lane 1: Untransfected control.

Lane 2: HEK293 cells transfected with DYKDDDDK-tagged protein vector

Dilution at 1:5000.

Recommended secondary antibody: Goat anti mouse IgG-HRP (Engibody, Catalog number:AT0098)

Recommended ECL: ECL Pico-Detect™ Western Chemiluminescent HRP Substrate (Engibody, Catalog number: IF6747)

Western Blot: DYKDDDDK Epitope Tag Antibody - Analysis of 30ug of whole cell lysates from HeLa cells infected with a strain of Chlamydia trachomatis expressing FLAG-IncA under the control of a Tetracycline promoter (Tet), and either left untreated (-) or treated with anhydrotetracycline (+), onto an SDS-PAGE gel.

Recommended secondary antibody: Goat anti mouse IgG-HRP (Engibody, Catalog number:AT0098)

Recommended ECL: ECL Pico-Detect™ Western Chemiluminescent HRP Substrate (Engibody, Catalog number: IF6747)

Western Blot: DYKDDDDK Epitope Tag Antibody

A. Untransfected 293T whole cell lysate/extract (30 ug)

B.-D. Whole cell lysate/extracts (30 ug) from transfected 293T cells expressing different DYKDDDDK-tagged proteins.

10% SDS-PAGE Anti-DYKDDDDK tag antibody dilution: 1:5000.

Recommended secondary antibody: Goat anti mouse IgG-HRP (Engibody, Catalog number:AT0098)

Recommended ECL: ECL Pico-Detect™ Western Chemiluminescent HRP Substrate (Engibody, Catalog number: IF6747)