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Purification & Detection

FLAG Antibodies

Detection / Purification of FLAG Fusion Proteins

The FLAG system uses three monoclonal ANTI-FLAG antibodies to detect and purify FLAG fusion proteins. While each recognizes and binds to the FLAG epitope, they exhibit different specificities that depend on the position of the FLAG peptide in the fusion protein. Use the Anti-FLAG® antibodies for immunoblotting, immunoprecipitation, ELISA and other immunodetection techniques.

Biotinylated ANTI-FLAG antibodies BioM2 and BioM5 are useful for enzyme-linked immunodetection (using streptavidin- or avidin-conjugated enzymes), and are necessary for detecting proteins expressed in mouse host cells.

Affinity gels (antibodies conjugated to agarose) are useful for affinity purification, and can be used repeatedly. Select the gel based on the position of the FLAG and the elution protocol you choose. ANTI-FLAG M1-agarose can be eluted with EDTA; both M1- and M2-agarose can be eluted with FLAG peptide or with glycine-HCl buffer, pH 3.5.

Pure FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) can be used to competitively displace FLAG-fusion proteins and elute them from FLAG affinity gels. This is a particularly mild elution, with negligible potential for damaging the fusion protein.

The table summarizes the components of the FLAG system that are used in detection and purification of fusion proteins.

 

Antibody & binding characteristics*

ANTI-FLAG M1

Mouse
IgG2b MAb

ANTI-FLAG M2

Mouse
IgG1 MAb

ANTI-FLAG
M5


Mouse
IgG1 MAb

Unprocessed N-terminal FLAG fusion protein:
Signal peptide - FLAG - Protein
- + Not determined
Met-N-terminal FLAG fusion protein:
Met - FLAG -Protein
- + ++
N-terminal FLAG fusion protein:
FLAG - Protein
+ + Weak
Internal FLAG peptide:
Protein - FLAG - Protein
- + Not determined
C-terminal FLAG fusion protein:
Protein - FLAG
- + Weak
Calcium-dependent binding
(complex is dissociated in EDTA-containing buffer)
+ - -
Comments Binds to N-term FLAG fusion proteins in a Ca++-dependent manner; useful for affinity purification Binds to FLAG epitope at any position infusion protein; can be dissociated by competition with FLAG peptide Best for detecting Met-FLAG peptides expression cytoplasm; binds too strongly to be useful in affinity purification

 

*Binding affinities: - no binding + binding ++ high affinity

 


Monoclonal ANTI-FLAG antibodies
Product Name Product #  Package sizes
ANTI-FLAG M1 F 3040 200 µg
1 mg
5 mg
ANTI-FLAG M2 F 3165 200 µg
1 mg
5 mg
ANTI-FLAG M5 F 4042 200 µg
1 mg
5 mg

 

Biotinylated ANTI-FLAG antibodies
Product Name Product #  Package sizes
ANTI-FLAG M1 NA  
ANTI-FLAG M2 F 9291 200 µg
1 mg
5x1 mg
ANTI-FLAG M5 F 2922 200 µg
1 mg

 


ANTI-FLAG affinity gels
Product Name Product #  Package sizes
ANTI-FLAG M1 A 1080 1 mL
5 mL
10 mL
25 mL
ANTI-FLAG M2 A 1205 1 mL
5 mL
10 mL
25 mL
ANTI-FLAG M5 NA  

 

FLAG peptide
Product Name Product #  Package sizes
ANTI-FLAG M1 F 3290 4 mg
25 mg
ANTI-FLAG M2 NA  
ANTI-FLAG M5 NA  

FLAG® and ANTI-FLAG® are registered trademarks of Sigma-Aldrich Co.The product designations, pFLAG™, pFLAG-1™, pFLAG-2™, FLAG-SHIFT™, pFLAG-SHIFT™, pFLAG-CTS™, pFLAG-ATS™, pFLAG-MAC™, pFLAG-CTC™, pFLAG-CMV™, YEpFLAG™ and FLAG-BAP™ are trademarks of Sigma-Aldrich Co. FLAG® products are manufactured by Sigma under license from Immunex Corp.

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