Nano-Booster FAQs

Frequently asked technical questions on GFP-and RFP-Booster


ChromoTek  Nano-Boosters  are ideal for Super-Resolution and “traditional” fluorescence microscopy because of their high affinity and extremely small size of just 2 to 3 nm. Technically speaking, the GFP-and RFP-Boosters are composed of the highly specific GFP- or RFP-binding domains of alpaca antibodies (also called “nanobodies”), covalently coupled to a selection of fluorescent dyes.

More and more customers use ChromoTek Nano-Boosters for both conventional immunofluorescence imaging and dSTORM, STED or 3D-SIM. Here we provide a list of answers to frequently asked technical questions that have been addressed to our technical support:

What GFP variants does the   GFP-Booster   recognize?

GFP-Booster specifically binds to most common GFP derivatives:

  • Cyan: eCFP, CFP, mCerulean
  • Green: eGFP, wtGFP, GFP S65T, AcGFP, TagGFP, tagGFP2, sfGFP, pHluorin, mClover
  • Yellow: eYFP, YFP, Venus, Citrine


What RFP variants does the  RFP-Booster   recognize?
  • mRFP, mCherry, mRFPruby, mPlum, DsRed


Do Nano-Boosters work on (methanol-)fixed samples?

Yes. Booster stainings perform equally well after fixation with the most common reagents: paraformaldehyde, glutaraldehyde, and methanol (Kaplan & Ewers, 2015; Ries et al., 2012).


Are Nano-Boosters applicable for live-cell imaging?

Yes, if the GFP/RFP-fusion is on the cell surface.

Being a 15 kDa protein, the Nano-Boosters do not penetrate the non-permeabilized cell membrane. Hence, if your GFP/RFP-fusion is intracellular, you have to fix and permeabilize the cells. Alternatively, it is possible to microinject or electroporate the Nano-Booster protein into the cells for live-cell imaging, but these approaches are rarely used.


What is the protocol for live-cell Nano-Booster staining of the extracellular GFP/RFP-fusions?

Incubate the cells with 1:25 Nano-Booster in growth media for 15 min at +4°C, wash, and image. This protocol will highlight just the plasma membrane pool of your GFP/RFP-fusion protein.


Do Nano-Boosters penetrate though the cell membranes of live cells?

No. Nano-Boosters are 15 kDa in size and therefore don’t penetrate through non-permeabilized cell membranes. If you need to deliver Nano-Booster into live cells, you may want to apply protein transduction methods (e.g. electroporation) or reagents, however from our experience, the most efficient way is to microinject the Boosters.


Can I do simultaneous co-stainings with GFP and RFP-Boosters?

Yes, you can combine the Nano-Boosters. For example, if you typically use the Nano-Boosters in a 1:200 dilution, you should add 1 µl each of gba488 and rba594 to 200 µL of blocking solution for a co-staining.


What Nano-Booster conjugates are recommended for super-resolution microscopy?

Nano-Boosters are highly suitable for Super-Resolution Microscopy. Due to their small size (2-3 nm), they minimize the linkage error and provide a more precise and dense staining, than conventional antibodies (15 nm linear dimension). The selection of a Nano-Booster conjugate depends on your microscope setup and lasers. We recommend for:

  • STED:    ATTO647N, Abberior STAR 635P
  • STORM: ATTO488/594/647N, or custom conjugation to Alexa647
  • SIM:       ATTO488/594


Is it possible to conjugate Nano-Boosters to other fluorophores?

Yes. You can label the ChromoTek GFP-Binding Protein (GFP nanobody; product code: gt-250) with NHS-activated fluorescent dyes following the instructions of the dye manufacturer.


How many dye molecules are coupled to Nano-Boosters?

Each Nano-Booster molecule carries on average one fluorophore (DOL = 0.8-1.2). Due to the “monoclonal”-like nature of Nano-Boosters, one molecule of a fluorescent protein can be bound by only one Nano-Booster molecule.


What is the concentration of Nano-Boosters?

The Nano-Booster concentration is in the range of 0.5 – 1 g/L. The concentration is adjusted for the best IF performance at 1:200 dilution, which is recommended in the product manual.


Can I do two-color Super-Resolution microscopy combining GFP- and RFP-Boosters?

Yes, dual-color STORM with Nano-Boosters is described in Bleck et al., PNAS 2014  and Platonova et al., ACS Chem Biol 2015


Can I do IF in yeast with Nano-Boosters?

Yes, immunostaining of yeast with Nano-Boosters is in fact simpler than with traditional (IgG) antibodies, because Nano-Boosters can penetrate the yeast cell wall due to their small size. For an optimized protocol for yeast staining with Nano-Boosters (here a GFP nanobody) see Kaplan & Ewers, Nat Protoc. 2015:


For what cell types and organisms can I use the Nano-Boosters to conduct IF experiments?

Nano-Boosters have been tested in cell culture, yeast, tissue slices, fly, zebrafish, and mouse. Tests in C. elegans have not been successful so far.


Do Nano-Boosters work in immunohistochemistry (IHC) on tissue sections? If so, what is a recommended protocol?

Yes, our customers successfully use GFP- and RFP-Boosters in IHC on both frozen and paraffin sections. For example, the following IHC conditions have been have published in

“For analysis of dendritic spines mice were anesthetized and transcardially perfused with PBS and then 4% para-formaldehyde/PBS. Brains were post-fixed in 4% PFA/PBS for 1 hour and 50μm sagittal sections cut using a Leica Vibratome. Floating brain sections were incubated for 2 hours in blocking solution (3 % Bovine Serum Albumin (BSA), 5 % Normal Goat Serum (NGS) and 0.2 % Triton X100 in PBS) and then incubated overnight with GFP booster Atto 488 (1:200 in 2 % BSA, 5 % NGS in PBS). Sections were then washed 4x20 mins in PBS and mounted on microscopy slides using Fluoromount (Sigma-Aldrich, Arklow, Ireland).”