RFP-Booster

Description
anti-RFP VHH/ Nanobody conjugated to fluorophore for Immunofluorescence of mCherry and other red fluorescent protein-fusion proteins

Conjugations
Alexa Fluor® 647
ATTO 594
ATTO 647N
Unconjugated

Specificity
mRFP, mCherry, mRFPruby, mPlum, DsRed

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 RFP-Booster Atto594 Size 10 µL Code rba594-10 Price $ 100
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Product RFP-Booster Atto594 Size 100 µL Code rba594-100 Price $ 365
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Product RFP-Booster Atto647N (please inquire) Size 10 µL Code rba647n-10 Price $ 110
Buy +
Product RFP-Booster Atto647N (please inquire) Size 100 µL Code rba647n-100 Price $ 395
Buy +
Product RFP-Booster Alexa Fluor® 647 Size 10 µL Code rb2AF647-10 Price $ 170
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Product RFP-Booster Alexa Fluor® 647 Size 50 µL Code rb2AF647-50 Price $ 395
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Product RFP VHH, recombinant binding protein Size 250 µL Code rt-250 Price $ 275
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Product RFP-Binding protein, custom labeling Size Code custom Price Please inquire

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

Specificity
DsRed, mCherry, mPlum, mRFP, mRFPruby

Host/isotype
Alpaca/recombinant VHH domain, monoclonal

Available conjugates
Alexa Fluor® 647, ATTO 594, ATTO 647N

Recommended dilution for RFP-Booster 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 RFP-Booster ATTO 594, ATTO 647N
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 or 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, 50 μL, 100 μL
Because of the small size of the VHH these volumes correspond to about 10 times the volume of conventional IgG antibodies.

Protein concentration
0.5 – 1 mg/mL (conjugates)

Storage instructions
Shipped at ambient temperature. Do not freeze. 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 5 nM

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 .

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.

Does the RFP-Booster recognize tdTomato?

No.

RFP-Booster ATTO647N or RFP-Booster Alexa Fluor® 647?

We currently offer our RFP-Booster conjugated to Alexa Fluor 647 and to ATTO647N. 
The RFP-Booster Alexa Fluor 647 has multiple advantages, when compared to RFP-Booster ATTO647N:

Improved RFP-Booster Alexa Fluor 647

RFP-Booster ATTO647N

Brighter fluorescence signal

About one third as bright

Site directed labelled

C-terminally 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 code: rb2AF647

Product code: rba647n

 

Note that we intend to replace RFP-Boosters ATTO 647N by RFP-Booster Alexa Fluor 647.

Please contact us if you require an ongoing supply of RFP-Booster conjugated to ATTO 647N. We are curious to learn why you need RFP-Booster ATTO647N and why you can’t use RFP-Booster Alexa Fluor 647 instead.

Stabilization, enhancement and reactivation of fluorescent protein signals with RFP-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 RFP-Booster will help you to get better images from your existing mCherry and other RFP expression constructs.

Benefits

  • Higher labeling 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!