Purification & Detection

FLAG® antibody selection: How to choose a detection antibody for FLAG-tagged proteins

 FLAG® antibody selection: How to choose a detection antibody for FLAG-tagged proteins

The FLAG® system uses three monoclonal anti-FLAG® antibodies to detect and purify FLAG®-tag proteins. While each antibody recognizes and binds to the FLAG® epitope (corresponding to the peptide sequence DYKDDDDK), they exhibit different specificities that depend on the position of the FLAG® peptide tag within the fusion protein. The FLAG® tag peptide can be incorporated into protein at the free amino-terminus (N-terminus), at the N-terminus as Met-N-terminal-FLAG®, at the carboxy-terminus (C-terminus), or internally. The anti-FLAG® M1 (M1 clone) antibody is highly specific for free N-terminal FLAG tag. The M2 antibody clone has strong affinity for FLAG tag at any position within the fusion protein. The M5 antibody clone strongly recognizes Met-N-FLAG® fusion protein, but binds weakly to free N-terminal or C-terminal FLAG® tags. Additionally, the anti-FLAG® antibody products are purified using different techniques, exhibit different sensitivities (as determined by dot blot analysis), and have been validated in different applications (including western blotting, immunoprecipitation, immunochemistry, and ELISA). Proper antibody selection will ensure optimal results for detection of a given FLAG®-tagged protein construct with a selected detection method.


Table 1: Antibody selection guidelines.

  Monoclonal anti-FLAG® M1 antibody produced in mouse Monoclonal anti-FLAG® M2
antibody produced in Mouse
Monoclonal anti-FLAG® M5 antibody produced in Mouse
Catalog Number F3040 F1804 F3165 F4042
Clone M1 M2 M2 M5
Antibody purification method Purified on Protein A Sepharose Purified on proprietary FLAG® peptide-agarose Purified on Protein A Sepharose Purified on Protein A Sepharose
KD 53.4 nM 9.3 nM 9.3 Nm Unavailable
Specificity Highly specific. Recognizes only the FLAG® epitope when located at the free N-terminus. Recognizes the FLAG® sequence at N-terminus, Met-N-terminus,
C-terminus or at any internal site of the FLAG® fusion protein.
Strong recognition of the Met-N FLAG® fusion protein. Weak binding with N- or C-terminal fusion protein.
Sensitivity Detects 1 ng of FLAG-BAP™ fusion protein on a dot blot Detects 2 ng of protein on a dot blot Detects <1 ng of Met-FLAG® fusion protein on a dot blot
Recommended for: Recommended for the detection of FLAG® fusion proteins in Mammalian, plants and bacterial expression systems. Very useful for affinity purification. Not recommended for detection of cytoplasmic expressed proteins. Western blot detection of target proteins expressed on E. coli,
plants or mammalian
crude cell lysate.
Western blot detection of target proteins expressed on mammalian and drosophilae cell lysate. Particularly useful for the detection of cytoplasmic expressed Met-FLAG fusion proteins. Not recommended for detection of fusion proteins expressed in E. coli
Applications* IP, IC, WB, EIA IP, IC, WB, EIA IP, IC, WB, EIA WB
Special requirements Binding requires calcium. (Complex will dissociate in the absence of calcium ions) N/A N/A N/A

*IP=immunoprecipitation; IC=immunochemistry, WB=western blot, EIA=enzyme-linked immunosorbent assay (ELISA)


Table 2: Binding characteristics of FLAG® antibodies

Antibody & binding characteristics*


IgG2b MAb


IgG1 MAb


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)
+ - -



Comparison of the specificity of M1 and two M2 antibodies


Western blots showing specificity of anti-FLAG M1 and M2 antibody clones for different tag positions

Figure 1: 5 µg of A431 cell lysate was spiked with 2 µg of N-terminal FLAG-BAP™ or C-terminal FLAG-BAP™ control protein and loaded in lane 2. The N-terminal FLAG BAP™ control protein was loaded in lane 3 and C-terminal FLAG BAP™ in lane 4. A control E.coli lysate with fusion FLAG® protein was loaded in lane 5. Standard immunodetection was performed using 2% NFDM for blocking. Primary antibodies were diluted 1:1,000 in blocking solution and incubated for 1 hr followed by 1 hr incubation of goat anti-mouse antibody diluted 1:50,000. Blots were detected using Immobilon® Forte HRP detection reagent.

Note that M1 antibody only recognizes the lanes where N-terminal FLAG® protein was present but the two M2 antibodies recognize all the lanes where N- or C-FLAG BAP™ protein was present.