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GFP and Epitopes  >  Antibodies to FLAG™ and Antibodies to 6XHIS Tags

Antibody for the detection of FLAG™ conjugated proteins

Rabbit Polyclonal
NCI Collaboration
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Rockland's antibody to detect FLAG™ conjugated proteins is shown to detect as little as 3 ng of amino-terminal FLAG™ tagged recombinant protein by western blot.  This antibody was used at a 1:1,000 dilution to detect 3-fold serial dilutions of amino-terminal FLAG™-Bacterial Alkaline Phosphatase (BAP) fusion protein (Sigma P-7582) starting at 1.0 µg of protein as shown in lanes 1-6 respectively.   A 4-20% gradient gel was used to separate the protein by SDS-PAGE.  The protein was transferred to nitrocellulose using standard methods.  After blocking, the membrane was probed with the primary antibody for 1 h at room temperature followed by washes and reaction with a 1:10,000 dilution of IRDye® 800 conjugated Gt-a-Rabbit IgG (H&L) (code 611-132-122) for 30 min at room temperature.  LICOR's Odyssey® Infrared Imaging System was used to scan and process the image.  Other detection systems will yield similar results
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250 µg
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Antibody for the detection of FLAG™ conjugated proteins Properties

Antibody for the detection of FLAG™ conjugated proteins (RABBIT) - 600-401-383
Known Cross Reactivity
Carboxy and amino terminal linked FLAG™ tagged recombinant proteins
ELISA : 1:20,000 - 1:40,000
Western Blot : 1:2,000 - 1:10,000
Immunohistochemistry: User Optimized
Other Dilution: User Optimized
Epitope Tag Type
Physical State
Liquid (sterile filtered)
Shipping Condition
Dry Ice
1.9mg/mL by UV absorbance at 280 nm
0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
0.01% (w/v) Sodium Azide
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Antibody for the detection of FLAG™ conjugated proteins Description

Epitope tags are short peptide sequences that are easily recognized by tag-specific antibodies.  Due to their small size, epitope tags do not affect the biochemical properties of the tagged protein.  Most often, sequences encoding the epitope tag are included with the target DNA at the time of cloning to produce fusion proteins containing the epitope tag sequence.  This allows Anti epitope tag antibodies to serve as universal detection reagents for any tag-containing protein produced by recombinant means.  This means that anti-epitope tag antibodies are a useful alternative to generating specific antibodies to identify, immunoprecipitate or immunoaffinity purify a recombinant protein.  The anti-epitope tag antibody is usually functional in a variety of antibody-dependent experimental procedures.
This antibody was purified from whole rabbit serum prepared by repeated immunizations with the Enterokinase Cleavage Site (ECS) peptide DYKDDDDK (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) conjugated to KLH using maleimide.  Residues of glycine and cysteine were added to the carboxy terminal end to facilitate coupling.  This antibody reacts with FLAG™ conjugated proteins.
Immunogen Type
Storage Condition
Store vial at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.
Application Note
This antibody is optimally suited for monitoring the expression of FLAG™ tagged fusion proteins.  As such, this antibody can be used to identify fusion proteins containing the FLAG™ epitope.  The antibody recognizes the epitope tag fused to either the amino- or carboxy- termini of targeted proteins.  This antibody has been tested by ELISA and western blotting against both the immunizing peptide and FLAGä containing recombinant proteins.  Although not tested, this antibody is likely functional for immunoprecipitation, immunocytochemistry, and other immunodetection techniques.  The epitope tag peptide sequence was first derived from the 11-amino-acid leader peptide of the gene-10 product from bacteriophage T7.  Now the most commonly used hydrophilic octapeptide is DYKDDDDK.  Rockland Immunochemical's polyclonal antibody to detect FLAG™ conjugated proteins binds FLAG™ containing fusion proteins with greater affinity than the widely used monoclonal M1, M2 and M5 clones, and shows greater sensitivity in most assays.  Affinity purification of the polyclonal antibody results in very low background levels in assays and low cross-reactivity with other cellular proteins.
This affinity purified antibody is directed against the FLAG™ motif and is useful in determining its presence in various assays. This polyclonal anti-FLAG™ tag antibody detects over-expressed proteins containing the FLAG™ epitope tag.  In western blotting of bacterial extracts, the antibody does not cross-react with endogenous proteins.
Disclaimer Note-General
This product is for research use only and is not intended for therapeutic or diagnostic applications. Please contact a technical service representative for more information. All products of animal origin manufactured by Rockland Immunochemicals are derived from starting materials of North American origin. Collection was performed in United States Department of Agriculture (USDA) inspected facilities and all materials have been inspected and certified to be free of disease and suitable for exportation. All properties listed are typical characteristics and are not specifications. All suggestions and data are offered in good faith but without guarantee as conditions and methods of use of our products are beyond our control. All claims must be made within 30 days following the date of delivery. The prospective user must determine the suitability of our materials before adopting them on a commercial scale. Suggested uses of our products are not recommendations to use our products in violation of any patent or as a license under any patent of Rockland Immunochemicals, Inc. If you require a commercial license to use this material and do not have one, then return this material, unopened to: Rockland Inc., P.O. BOX 5199, Limerick, Pennsylvania, USA.
General Reference
Chubet, R.G. and Brizzard, B.L. (1996) Vectors for expression and secretion of FLAG epitope-tagged proteins in mammalian cells. Biotechniques 20(1):136-141. Slootstra, J.W., et al. (1997) Identification of new tag sequences with differential and selective recognition properties for the anti-FLAG monoclonal antibodies M1, M2 and M5. Mol. Divers. 2(3):156-164. Robeva, A.S., et al. (1996) Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope. Development of an efficient generic protein purification procedure. J. Biochem. Pharmacol. 51(4):545-555. Fulton, J.E. et al. (1995) Functional analysis of avian class I (BFIV) glycoproteins by epitope tagging and mutagenesis in vitro. Eur. J. Immunol. 25(7):2069-2076.
Specific Reference
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Product Type
Primary Antibodies;
Reacts With
human, mouse, rat, monkey
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Secondary Antibodies;
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Secondary Antibodies;
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Supporting Reagents;
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Conjugation Reference
Molecular Weight
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Conjugation Chemistry