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 http://www.ncbi.nlm.nih.gov/pubmed/25099357 and Platonova et al., ACS Chem Biol 2015 http://www.ncbi.nlm.nih.gov/pubmed/25806422
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: www.ncbi.nlm.nih.gov/pubmed/26068895
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 http://www.ncbi.nlm.nih.gov/pubmed/24035894:
“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).”