GFP-Booster

Description
anti-GFP VHH/ Nanobody conjugated to a fluorescent dye for IF/ microscopy.

Conjugations
Alexa Fluor® 647
ATTO488
ATTO594
ATTO647N
Abberior STAR635P
Unconjugated

Specificity
eCFP, CFP, mCerulean, eGFP, wtGFP, GFP S65T, AcGFP, TagGFP, tagGFP2, mClover (Clover A206K), sfGFP, pHluorin, eYFP, YFP, Venus, Citrine

Applications
Immunofluorescence (IF): Immunohistochemistry (IHC), Immunocytochemistry (ICC)
Wide-field fluorescence and confocal microscopy, super-resolution microscopy (SRM), light-sheet microscopy
Cleared tissue, organ, and animal imaging

ProductSizeCodePriceBuy
ProductGFP-Booster Atto488Size10 µLCodegba488-10Price $ 100
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ProductGFP-Booster Atto488Size100 µLCodegba488-100Price $ 365
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ProductGFP-Booster Atto594Size10 µLCodegba594-10Price $ 100
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ProductGFP-Booster Atto594Size100 µLCodegba594-100Price $ 365
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ProductGFP-Booster Atto647NSize10 µLCodegba647n-10Price $ 110
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ProductGFP-Booster Atto647NSize100 µLCodegba647n-100Price $ 395
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ProductGFP-Booster Alexa Fluor® 647Size10 µLCodegbAF647-10Price $ 110
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ProductGFP-Booster Alexa Fluor® 647Size100 µLCodegbAF647-100Price $ 395
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ProductGFP-Booster Abberior AS 635PSize10 µLCodegbas635p-10Price $ 100
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ProductGFP-Booster Abberior AS 635PSize100 µLCodegbas635p-100Price $ 365
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ProductGFP VHH, recombinant binding proteinSize250 µLCodegt-250Price $ 275
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ProductGFP-Binding protein, custom labelingSizeCodecustomPrice Please inquire

Coupled Nanobody/ VHH
Recombinant, monoclonal anti-Green Fluorescent Protein (GFP) single domain antibody (sdAb) fragment

Specificity
AcGFP, Citrine, CFP, eCFP, eGFP, eYFP, GFP S65T, mCerulean, pHluorin, sfGFP, mClover (Clover A206K), TagGFP, tagGFP2, Venus, wtGFP, YFP

Host/ isotype
Alpaca/recombinant VHH domain, monoclonal

Available conjugates
ATTO488, ATTO594, ATTO647N, Abberior STAR635P, unconjugated

Recommended dilution
IF/ICC/IHC: 1:200 Optimal working concentration is application-dependent and should be determined by testing a range of dilutions from 1:50 to 1:1,600

Microscopy techniques
Wide-field epifluorescence microscopy; confocal microscopy; super-resolution microscopy e.g. 3D-SIM, PALM, STED, STORM; light-sheet microscopy

Form
Purified recombinant protein in PBS supplemented with preservative 0.09 % sodium azide

Size
10 μL, 100 μL
Because of the small size of a VHH these volumes contain about 10 times more molecules than  a comparable volume of conventional IgG antibodies.

Protein concentration
0.5 – 1 mg/mL (conjugates)

Storage instructions
Shipped at ambient temperature. Upon receipt store at 4°C. Do not freeze. Protect from light.

Validation
Validated in cell culture & cell lines, tissue sections, yeast, fly, zebrafish, mouse, in germline of Caenorhabditis elegans, in Drosophila melanogaster embryos and cleared whole mouse. Fixed cultured cells: formaldehyde, methanol or glutaraldehyde fixation
Tissue sections: cryosections, FFPE paraffin sections
Whole specimen: cleared mouse

Affinity
Dissociation constant KD of 1 pM, detected using switchSENSE® technology from Dynamic Biosensors

Which Nano-Booster and Nano-Label conjugates are recommended for super-resolution microscopy?

Nano-Boosters and Nano-Labels 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 and Nano-Label conjugate depends on your microscope setup and lasers. We recommend for:
- STED: ATTO647N, Abberior STAR 635P
- STORM: Alexa Fluor 647, ATTO488
- SIM: ATTO488/594

Are Nano-Booster applicable for live-cell imaging?

Yes, if the fusion-tag is on the cell surface.
Nano-Boosters and Nano-Labels are small proteins and therefore don’t penetrate through non-permeabilized cell membranes. Hence, if your fusion-protein is intracellular, 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 Nano-Boosters and Nano-Labels.

Can I do a simultaneous co-staining with two or more Nano-Boosters and Nano-Labels?

Yes, you can combine the Nano-Boosters and Nano-Labels. 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.

How many dye molecules are coupled to Nano-Boosters and Nano-Labels?

Each Nano-Booster and Nano-Label molecule carries on average 1-2 fluorophores.
Note: Nano-Boosters labeled with Atto647N carry a maximum of 1 fluorophore per VHH.

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 .

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

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

What is the protocol for live-cell Nano-Booster and Nano-Label staining of the extracellular fusion protein?

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

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

No. Nano-Boosters and Nano-Labels are small proteins and therefore don’t penetrate through non-permeabilized cell membranes. If you need to deliver Nano-Booster and Nano-Labels 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 Nano-Boosters and Nano-Labels.

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

Yes. You can label the ChromoTek GFP-Binding Protein (GFP VHH, product code: gt-250), RFP-Binding Protein (RFP VHH, product code: rt-250) and Spot-Binding Protein (Spot VHH, product code: etx-250) with NHS-activated fluorescent dyes following the instructions of the dye manufacturer.
Note: Spot VHH contains a sortase-tag at its C terminus (sortase recognition motif LPETG) which can be used for conjugation.

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.

Stabilization, enhancement and reactivation of fluorescent protein signals with GFP-Booster

Fluorescent proteins (FPs) are powerful tools to study protein localization and dynamics in living cells. However, genetically encoded FPs have several disadvantages compared to chemical dyes:

  • Signal intensities of fixed samples from cells expressing FP fusion at physiological expression levels are usually very low.
  • Both photostability and quantum efficiency of FPs are generally not sufficient for super-resolution microscopy (e.g. 3D-SIM, STED or STORM/PALM).
  • Many cell biological methods such as HCl treatment for BrdU-detection, the EdU-Click-iT™ treatment or heat denaturation for FiSH lead to disruption of the FP signal.

Here, the ChromoTek GFP-Booster will help you to get better images from your existing GFP expression constructs.

 

Benefits

  • Higher labelling density
  • Considerably higher tissue penetration rates
  • Superior accessibility and labelling of epitopes in crowded cellular/organelle environments
  • Less than 2 nm epitope-label displacement minimizes linkage error
  • Monovalent VHHs do not cluster their epitopes
  • Validation: structure and function characterized
  • Consistent and reliable performance due to recombinant production

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Only for research applications, not for diagnostic or therapeutic use!