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GFP-Booster

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STED microscopy of Vimentin filaments. Vero cells expressing Vimentine-Citrine were immunostained with GFP-Booster (image kindly provided by F. Winter, dept. NanoBiophotonics, MPI-bpc, Goettingen).
Confocal image of HeLa cells transiently transfected with Tom70-eGFP and immunostained with GFP-Booster Alexa Fluor 488 (green). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich
Tissue penetration rate:
The comparison of conventional anti-GFP antibody and GFP-Booster shows the superior tissue penetration rate of GFP-Booster. Fluorescent images of transgenic mouse tissue expressing Cx3Cr1-EGFP. EGFP signal was enhanced either with conventional anti-GFP antibody (top image) or with the GFP-Booster (bottom image).
Confocal images of HeLa cells transiently transfected with MannosidaseII-GFP (green) and immunostained with GFP-Booster Alexa Fluor 568 (red). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
Immunostaining of Tubulin-GFP: HeLa cells low expressing Tubulin-GFP. Top: GFP signal of Tubulin-GFP (green) and DAPI stain (blue); Bottom: Tubulin-GFP detection by GFP-Booster coupled to Alexa Fluor 647
Central nervous system of a 3-day-old Drosophila larvae. GFP-Booster was used to enhance the signal of dopamine neurons expressing GFP. (Image kindly provided by Kayvan Forouhesh Tehrani, the Kner Lab, University of Georgia; the Drosophila sample was supplied by the Shen lab, University of Georgia, Athens.)
Enhancement of GFP signal with GFP-Booster Atto488: Comparison of signal intensity of a cell line stably expressing a nuclear GFP-fusion protein before and after GFP-Booster treatment.

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

Conjugations
Alexa Fluor® 488
Alexa Fluor® 568
Alexa Fluor® 647
ATTO488
ATTO594
ATTO647N
Unconjugated

Specificity
CFP, eGFP, AcGFP, mClover (Clover A206K), eYFP, Venus and more GFP derivatives, see:
Fluorescent protein specificity table Nano-Booster

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

Product Size Code Price Buy
Product GFP-Booster Alexa Fluor® 488 Size 10 µL Code gb2AF488-10 Price $ 170
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Product GFP-Booster Alexa Fluor® 488 Size 50 µL Code gb2AF488-50 Price $ 395
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Product GFP-Booster Alexa Fluor® 568 Size 10 µL Code gb2AF568-10 Price $ 170
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Product GFP-Booster Alexa Fluor® 568 Size 50 µL Code gb2AF568-50 Price $ 395
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Product GFP-Booster Alexa Fluor® 647 Size 10 µL Code gb2AF647-10 Price $ 170
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Product GFP-Booster Alexa Fluor® 647 Size 50 µL Code gb2AF647-50 Price $ 395
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Product GFP-Booster Atto488 Size 10 µL Code gba488-10 Price $ 100
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Product GFP-Booster Atto488 Size 100 µL Code gba488-100 Price $ 365
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Product GFP-Booster Atto594 Size 10 µL Code gba594-10 Price $ 100
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Product GFP-Booster Atto594 Size 100 µL Code gba594-100 Price $ 365
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Product GFP-Booster Atto647N Size 10 µL Code gba647n-10 Price $ 110
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Product GFP-Booster Atto647N Size 100 µL Code gba647n-100 Price $ 395
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Product GFP VHH, recombinant binding protein Size 250 µL Code gt-250 Price $ 275
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Product GFP-Binding protein, custom labeling Size Code custom Price Please inquire

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

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

Host/ isotype
Alpaca/recombinant VHH domain, monoclonal

Available conjugates
Alexa Fluor® 488, Alexa Fluor® 568, Alexa Fluor® 647, ATTO488, ATTO594, ATTO647N, unconjugated

Recommended dilution for GFP-Booster Alexa Fluor® 488, Alexa Fluor® 568, Alexa Fluor® 647
IF/ICC/IHC: 1:500- 1:1,000 Optimal working concentration is application-dependent and should be determined by testing a range of dilutions from 1:100 to 1:4,000

Recommended dilution for GFP-Booster ATTO488, ATTO594, ATTO647N
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, 50µL, 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)

RRID
GFP-Booster Alexa Fluor 488: AB_2827573
GFP-Booster Alexa Fluor 568: AB_2827574
GFP-Booster Alexa Fluor 647: AB_2827575
GFP-Booster Atto488: AB_2631386
GFP-Booster Atto594: AB_2631387
GFP-Booster Atto647N: AB_2629215
GFP-Booster Abberior AS 635P: AB_2631388

Storage instructions
Shipped at ambient temperature. Protect from light. For storage instructions see Manual/ Data Sheet. 

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. Nano-Boosters conjugated to Alexa Fluor® dyes are labeled in a site-directed way and carry in total 2 fluorophores per VHH. Nano-Boosters labeled with Atto647N carry a maximum of 1 fluorophore per VHH at the C-terminus.

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: etb-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.

GFP-Booster Alexa Fluor or GFP-Booster ATTO

We currently offer our GFP-Booster conjugated to different Alexa Fluor dyes and to several ATTO fluorophores. 
The GFP-Boosters conjugated to Alexa Fluor have multiple advantages, when compared to GFP-Boosters labelled with ATTO dyes:

 

Improved GFP-Booster Alexa Fluor

GFP-Booster ATTO

Brighter fluorescence signal

About one third as bright

Site directed labelled

Randomly labelled

Degree of labeling DOL = 2 dyes per VHH

Labelled with average 1 fluorophore per VHH

Dilution range: 1:500 – 1:1,000

Dilution range: 1:200

Product codes: gb2AF488, gb2AF568, gb2AF647

Product codes: gba488, gba594, gba647n

 

Note that we intend to replace GFP-Boosters ATTO by GFP-Booster Alexa Fluor.

Please contact us if you require an ongoing supply of GFP-Boosters conjugated to ATTO dyes. We are curious to learn why you need GFP-Booster ATTO and why you can’t use GFP-Boosters Alexa Fluor instead.

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

  • 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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