anti-RFP VHH/ Nanobody conjugated to fluorophore for Immunofluorescence of mCherry and other red fluorescent protein-fusion proteins
Alexa Fluor® 647
mRFP, mCherry, mRFPruby, mPlum, DsRed
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 RFP-Booster Atto594||Size 10 µL||Code rba594-10||Price $ 100||Buy +|
|Product RFP-Booster Atto594||Size 100 µL||Code rba594-100||Price $ 365||Buy +|
|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||Buy +|
|Product RFP-Booster Alexa Fluor® 647||Size 50 µL||Code rb2AF647-50||Price $ 395||Buy +|
|Product RFP VHH, recombinant binding protein||Size 250 µL||Code rt-250||Price $ 275||Buy +|
|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
DsRed, mCherry, mPlum, mRFP, mRFPruby
Alpaca/recombinant VHH domain, monoclonal
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
Wide-field or epifluorescence microscopy; confocal microscopy; super-resolution microscopy e.g. 3D-SIM, PALM, STED, STORM, light sheet microscopy
Purified recombinant protein in PBS supplemented with preservative 0.09 % sodium azide
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.
0.5 – 1 mg/mL (conjugates)
Shipped at ambient temperature. Do not freeze. Protect from light. For storage instructions see Manual/ Data Sheet.
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
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?
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?
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
Brighter fluorescence signal
About one third as bright
Site directed 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.
- 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
Only for research applications, not for diagnostic or therapeutic use!