Anti-Tag antibodies you can rely on: a focus on GFP and RFP

My colleague Ana introduced in a previous post (3 years ago already!) a selection of the most popular anti-tag antibodies among the research community. I must say that they remain extremely popular, showing their robustness over time. In this post, I would like to focus on anti-RFP antibodies and anti-GFP antibodies, looking at a selection of high quality, fully validated, reliable tools.

Green fluorescent protein (GFP) and Red fluorescent protein (RFP) are ~29 kDa fluorescent proteins used as versatile markers for visualization of protein localization, monitoring physiological processes and for detecting transgenic or transient protein expression. They can be used for the generation of fusion proteins without significantly interfering with native protein assembly and function, making them powerful tools for in vivo analyses. Antibodies against such tags are very likely to be used for years in the same lab (at least for longer periods than target specific antibodies), hence they require higher batch to batch reproducibility over time. They are also likely to be used by several researchers in the same lab, possibly on different applications. Finally, as there are so many on the market, the choice is not always easy. Well, it’s my task to suggest ones you can rely on – as I have selected for you below!

Based on our experience and feedback from satisfied users, we have selected those produced by Rockland, a US based antibody manufacturer since 56 years, using the highest standards and procedures (GMP compliant; TRACES approved and USDA licensed), a guarantee for reliability and robustness.

Anti-GFP antibodies

Rockland anti GFP antibodies are suitable for the detection of native GFP, GFP variants such as S65T-GFP, RS-GFP, YFP and EGFP and most other GFP fusion proteins by ELISA, Flow Cytometry, Immunofluorescent Microscopy, Immunohistochemistry, Immunoprecipitation and western blot. Both monoclonal and polyclonal antibodies are available, raised against full length GFP, with several conjugated formats, from Peroxidase (HRP) and BIOTIN, to fluorescent dyes such as Atto Dye, FITC, and DyLight Dye. You can screen the list of available formats at this link. A special note for the TrueBlot® Immunoprecipitation and Western Blot Kit for GFP Epitope Tag. It allows for the immunoprecipitation and detection of GFP-tagged recombinant proteins present in cell lysates. The very clean signal obtained with the kit is illustrated in the right hand side picture below.

Anti-GFP (Rabbit) antibody
Fluorescein conjugated (600-402-215)

TrueBlot and WB kit for
GFP Epitope tag (88-8887-215)

Anti-RFP antibody selection

All Rockland RFP antibodies are affinity-purified assuring both high affinity and specificity and have been pre-absorbed to eliminate any potential cross-reactivity to human, mouse and rat serum proteins. The antibodies are also confirmed for non-reactivity to GFP protein. In addition, conjugation of RFP antibodies to either fluorescent dyes, biotin or horseradish peroxidase are available to further facilitate RFP protein detection and quantification. The star among all these references is the rabbit polyclonal 600-401-379, validated in IHC, ELISA, WB, IP. I have selected a few of the numerous illustrations available for this very popular antibody.

Immunofluorescence Microscopy of Rabbit Anti-RFP antibody. Tissue: Hop derived cells frm ERCre/+; R26Tom/+ mice. Fixation: 0.5% PFA. Antigen retrieval: Tamoxifen. Primary antibody: RFP antibody at 10 µg/mL for 1 h at RT. Secondary antibody: Fluorescein rabbit secondary antibody at 1:10,000 for 45 min at RT. Localization: RFP is nuclear and occasionally cytoplasmic. Staining: Hop-derived cells in the hair follicle, labeled in red.

Immunofluorescence Microscopy of Rabbit Anti-RFP antibody. Tissue: DsRed transgenic mouse retina. Fixation: 4% PFA. Blocking: 3% BSA, 0.3% Triton Primary antibody: RFP antibody at 1:100 for 12 h at 4°C. Secondary antibody: Alexa488 secondary antibody at 1:10,000 for 4 hours at RT. Localization: RFP is nuclear and occasionally cytoplasmic. Staining: labeled in red.

Immunohistochemistry of Anti-RFP Antibody. Tissue: Mouse gut tissue in tomato transgenic mice. Fixation: formalin fixed paraffin embedded. Antigen retrieval: heat 5 min at high temp in 1X rodent decloaker. Primary antibody: RFP antibody at 1:200 for 1 h 30 min at RT. Secondary Antibody: HRP anti-rabbit (p/n 611-103-122). Image courtesy: Kwan Hyun Kim.

Now, if you haven’t found the antibody suited to your application among this selection, please feel free to contact me directly and I’ll be happy to help. Also for other anti-tag antibodies like anti-KLH, c-Myc, 6xHis, FLAG and many more… This convenient epitope tag sampler kit can also help you choose at a reasonable cost.

Isabelle Nobiron
Written by Isabelle Nobiron
Isabelle is a Product Manager at tebu-bio, and also the company's ISO Quality Manager.