A high-affinity monoclonal antibody against the FLAG tag useful for G-protein-coupled receptor study

doi:10.1016/j.ab.2012.03.014

Analytical Biochemistry

Volume 425, Issue 2, 15 June 2012, Pages 157-165

https://doi.org/10.1016/j.ab.2012.03.014 Get rights and content

Abstract

The FLAG sequence (DYKDDDDK) is an artificial sequence widely used to detect, quantify, and purify proteins expressed as FLAG–fusion proteins. Several highly specific monoclonal antibodies for FLAG are commercially available; however, they are not always sensitive enough to detect proteins expressed at low levels and can give rise to unacceptable levels of background signal when used for immunostaining in vitro and in vivo. The current study reports the successful establishment of hybridoma cells that produce an extremely high-affinity antibody to FLAG, namely 2H8 Ab. 2H8 Ab stained FLAG-tagged G-protein-coupled receptors more strongly than commercially available antibodies in both flow cytometry and immunostaining experiments with no background staining. 2H8 was sensitive enough to detect FLAG-tagged G-protein-coupled receptors and soluble proteins in crude preparations, which could not be achieved using commercially available antibodies. Only 10 ng of 2H8 Ab was required to immunoprecipitate FLAG-tagged G-protein-coupled receptors from cell lysates. Of note, 2H8 stained FLAG-tagged BLT2, a low-affinity leukotriene B₄ receptor, expressed in vivo in the small intestine of mice under control of the villin promoter. Thus, 2H8 Ab is a promising tool for analyzing various FLAG–fusion proteins, particularly G-protein-coupled receptors, both in vitro and in vivo.

Section snippets

Materials

Mouse monoclonal anti-FLAG mAbs (M2 and M5) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Alexa Fluor 488-conjugated anti-mouse immunoglobulin G Ab (anti-mouse IgG–Alexa 488) was purchased from Invitrogen (Carlsbad, CA, USA).

Animal studies

All animal experiments were performed in compliance with the standards established by the International Guiding Principles for Biomedical Research Involving Animals and were approved by the animal study committee of Kyushu University.

Establishment of anti-FLAG mAb

The anti-FLAG mAb was

Establishment of hybridoma (2H8)

The hybridoma cell line 2H8, which produces a high-affinity anti-FLAG Ab, was unexpectedly established during an attempt to generate an anti-mouse BLT1 mAb. Mice were immunized with NIH-3T3 cells stably expressing FLAG–mBLT1, and approximately 2000 hybridoma lines were established by fusing lymph node cells and splenocytes with SP2 myeloma cells. FACS screening identified clone 2H8, whose culture supernatant (sup) positively stained FLAG–mBLT1 expressing CHO cells (CHO–FLAG–mBLT1; data not

Discussion

Epitope tagging is one of the most widely used techniques in modern cell biology and biochemistry. FLAG, HA, and c-myc tags are the most common ones because high-affinity antibodies to these tags are commercially available. Tags are useful tools for detecting proteins for which specific Abs are not available because recent advances in molecular biology have made it possible to generate fusion proteins by fusing epitope tags to the target proteins. mAbs against these epitope tags are stable and

Conclusion

Epitope tagging is an important technique in biochemistry, but conventional anti-FLAG antibodies lack sensitivity and produce background staining. We established a novel anti-FLAG monoclonal antibody, 2H8, with a high sensitivity and low background. 2H8 is an optimal antibody for the detection of FLAG tags fused to the amino termini of various proteins. 2H8 will be a useful experimental tool in many areas of biological research.

Acknowledgments

We thank the Support Center for Education and Research (Kyushu University), Chikara Meno and Keiko Kitajima (Kyushu University) for technical support, Junichi Miyazaki (Osaka University) for supplying pCXN2, and all of our laboratory members for valuable advice and discussions. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (21390083, 22116001, and 22116002 to T.Y. and 20790232 and 22790296 to

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A high-affinity monoclonal antibody against the FLAG tag useful for G-protein-coupled receptor study

Abstract

Section snippets

Materials

Animal studies

Establishment of anti-FLAG mAb

Establishment of hybridoma (2H8)

Discussion

Conclusion

Acknowledgments

Cell

Cell

Cell

J. Biol. Chem.

Trends Biochem. Sci.

J. Immunol. Methods

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Epitope tagging

Annu. Rev. Genet.

A short polypeptide marker sequence useful for recombinant protein identification and purification

Nat. Biotechnol.