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