Anti-FLAG Epitope Tag Mouse Monoclonal Antibody (DBOA10007) DBOA10007

Anti-FLAG Epitope Tag Mouse Monoclonal Antibody (DBOA10007) Techniques DBio DBOA10007
Supplier: DBio
Product Code: DBOA10007
Availability: In Stock

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Anti-FLAG Epitope Tag Mouse Monoclonal Antibody (DBOA10007), 100 ug

The antibody recognizes the FLAG epitope in FLAG-tagged fusion proteins at the N-terminus or C-terminus.

Background

A fusion tag called FLAG and consisting of eight amino acids Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys including an enterokinase-cleavage site, was specifically designed for immunoaffinity chromatography. It allows elution under non-denaturing conditions. Several antibodies against this peptide have been developed.

The added marker segment is that it should not interfere with the native folding of proteins to which it is attached. Secondly, the marker peptide sequence should be water-soluble and should retain a high degree of exposure on the surface of the protein, so that it can readily interact with its ligand. It should also be suitable for a mild and inexpensive affinity purification procedure. Finally, an easy removal of the marker peptide leading to a native product is also advantageous. Due to this small size, the marker peptide can be encoded by a single synthetic oligonucleotide. It is known that aromatic amino acids are the major factors in antigen–antibody interactions. Lys at position 3 in the marker sequence leads to a hexapeptide sequence LysAspAspAspAspLys, which ensures a maximum value on the hydrophilicity scale according to Hopp and Woods2 . Such hydrophilic sequences have been shown to express strong antigenicity and are thus likely to adopt a highly exposed conformation in the three-dimensional folding of proteins3. Another virtue of FLAG is that the longest trypsinogen prosequences are of this length. This enables the removal of the tag and the production of an authentic N-terminus of the fusion protein partner by enterokinase treatment. The FLAG peptide can be fused to either the N- or C-terminus of a given fusion protein. Nevertheless, the N-terminal fusion has several advantages. Inhibition ELISA experiments showed that the anti-Flag antibody M1 binds three to four orders of magnitude better under conditions where the a-amino group of the first amino acid is freely accessible4. The FLAG marker peptide fusion system comprises a unique and widely useful technique for protein identification and purification. Elution of the fusion protein can be accomplished either by antibody-mediated affinity chromatography in a calcium-dependent manner, by lowering the pH, or by competitive elution with synthetic peptides1. Although highly selective, the binding capacities are low, making scale-up a costly undertaking. In addition to cost and low capacity, large-scale immunoaffinity chromatography, applied to the production of therapeutic proteins has several disadvantages: ligand leakage, instability, and need for validation of antibody production. The stability of the affinity chromatography column depends on the nature and source of the crude extracts. Furthermore, the FLAGe tag, designed to be immunogenic, must be removed from therapeutic proteins. In most cases, this can be accomplished with enterokinase. However, contaminating proteases may also produce undesired cleavages. Despite these drawbacks, the FLAG fusion is useful in research and development: FLAG proteins can be readily purified and assayed by ELISA or any other immunochemical detection method, thus expediting the raising of antisera against a desired protein and characterization studies.

References:

1. A. Einhauer, A. Jungbauer. The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J. Biochem. Biophys. Methods 49 2001 455–465.

2. Hopp TP, Woods KR. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A 1981;78:3824–8.

3. Hopp TP. Protein surface analysis: methods for identifying antigenic determinants and other interaction sites. J Immunol Methods 1986;88:1–18.

4. Power BE, Ivancic N, Harley VR, Webster RG, Kortt AA, Irving RA, et al. High-level temperature-induced synthesis of an a

Datasheet

Overview

 

Product Name  

Anti-FLAG Epitope Tag Mouse Monoclonal Antibody (DBOA10007), 100 ug

Description

Mouse Monoclonal Antibody (TBD), against FLAG  Epitope Tag, can be used in identify FLAG tagged fusion protein in Western blotting, immunoprecipitation immunofluorescence microscopy or other immunoassays.

Tested Applications

WB, IHC

Species Reactivities

Reacts with: FLAG tag

Immunogen     

FLAG tag peptide

Positive Control

N/A

 

Properties

 

Form 

Freeze dried solid

Storage Instructions

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

Storage Buffer              

When reconstituted with 100 deionized water, PBS with no preservatives.

Constituent

PBS

Purity  

Purified by protein A

Clonality

Monoclonal

Clone Name

F1L

Isotype

IgG1

Applications

The application notes include recommended starting dilutions; optimal dilutions or concentrations should be determined by the end user.

WB

1/500 - 1/2000.

IHC

1/200 - 1/1000

General
Applications WB, IP, IF
Concentration 1 mg/ml when reconstitute in 100 ul
Purity Purified from ascites
Size 100 µg
Source Species Mouse
Antibody
Immunogen DYKDDDDK, H-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-OH
Clonality Monoclonal
Isotype IgG1
Form Freeze dry solid
Storage Instructions Store at 4°C.
Storage Buffer Preservative: None; Constituents: PBS (without Mg2+ and Ca2+), pH 7.4, 150 mM NaCl, when reconstitute with 100 ul di water

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