Immunostaining of PFA fixed 3T3 cells expressing a TOM70-mCherry reporter protein with FluoTag®-X4 Atto 647N anti-RFP sdAb (Cat. No. N0404, dilution 1:500, the mCherry signal is represented in red, the corresponding FluoTag-signal is represented in green and the merge of both channels is represented in yellow). Nuclei were visualized by DAPI staining (blue).

A single motoneuron in the Drosophila larval central nervous system, fixed with 2% PFA. Cell membrane (RFP,red) and synaptic connections (YPet, blue), were stained with FluoTag-X4 anti-RFP (N0404-AF568-L) and FluoTag-X4 anti-GFP (N0304-AT488).

Image credit: Tom Pettini, Matthias Landgraf Lab, University of Cambridge, UK

FluoTag-X4 anti-RFP AF647

PFA-fixed Cos7 cells expressing a TOM70-nfRFP-BFP fusion protein (nf: non-fluorescent) were stained with FluoTag®-X4 anti-RFP coupled to Alexa Fluor 647 (Cat. No. N0404-AF647, dilution 1:500). A Greyscale image of the staining performed with N0404-AF647. B False color representation of the image shown in A is displayed in magenta (coloring according to the excitation wavelength of the employed fluorophore). C The corresponding DAPI signal of the depicted section. D Merge of A and C. False color representation of A in magenta and C in blue.

FluoTag-X4 anti-RFP AF568

PFA-fixed Cos7 cells expressing a TOM70-nfRFP-BFP fusion protein (nf: non-fluorescent) were stained with FluoTag®-X4 anti-RFP coupled to AZDye 568 (Cat. No. N0404-AF568, dilution 1:500). A Greyscale image of the staining performed with N0404-AF568. B False color representation of the image shown in A is displayed in red (coloring according to the excitation wavelength of the employed fluorophore). C The corresponding DAPI signal of the depicted section. D Merge of A and C. False color representation of A in red and C in blue.

FluoTag-X4 anti-RFP At488

PFA-fixed Cos7 cells expressing a TOM70-nfRFP-BFP fusion protein (nf: non-fluorescent) were stained with FluoTag®-X4 anti-RFP coupled to Atto 488 (Cat. No. N0404-At488, dilution 1:500). A Greyscale image of the staining performed with N0404-At488. B False color representation of the image shown in A is displayed in green (coloring according to the excitation wavelength of the employed fluorophore). C The corresponding DAPI signal of the depicted section. D Merge of A and C. False color representation of A in green and C in blue.

FluoTag®-X4 anti-RFP

Cat No: N0404 Category:

400,00 

FluoTag®-X4 anti-RFP is a blend of two in-house developed single-domain antibodies (sdAbs) recognizing the most common red fluorescent proteins like mRFP, mCherry, DsRed, and other DsRed derivatives with high affinity and specificity.

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One of the first red-emitting fluorescent proteins described was DsRed from Discosoma sp. After a couple of years from its publication in 1999 and more than 20 mutations, a monomeric version with several enhanced characteristics appeared as mRFP1. From this promising monomeric red-FP, dozens of monomeric variants have been described, being mCherry one of the most popular derivatives. Today more than 800 entries of various fluorescent proteins can be found in an open-source database with the most currently available variants in the fluorescence protein database “fpbase”

Our nanobodies bind specifically and strongly to DsRed, RFP, and mCherry, among other family members. Look at our specificity chart in the resource section.

FluoTags are directly conjugated to fluorophores, however, they can be equipped with a single fluorophore for more quantitative readouts (FluoTag-Q), with two fluorophores per single-domain antibody (FluoTag-X2), and we also developed a blend of two sdAbs bindings simultaneously the target proteins and each bearing two fluorophores, decorating the target protein with 4 fluorophores in total (FluoTag-X4). For more detailed information on the FluoTags, please check our Technology Section.

Variations:
Conjugation Amount Cat No. RRID
Atto488 200 μl N0404-At488-L AB_2744637
AZDye568 200 μl N0404-AF568-L AB_3075920
Atto643 200 μl N0404-At643-L AB_3075923
Alexa647 200 μl N0404-AF647-L AB_2905526
AbberiorStar635P 200 μl N0404-Ab635P-L AB_2744636
Related Products: -
Clone: 2B12, 2A1
Host: Alpaca
Produced in: E.coli
Application: IF
Dilution: 1:250 (corresponding to 5 nM for each sdAb clone)
Capacity: N/A
Antigen: -
Targets: mRFP
Specificity: mRFP (red fluorescent protein) and other derivatives like mOrange, dsRed1, dsRED2, tdTomato, mCherry and mScarlet-i.
Formulation: A mixture of two sdAb clones lyophilized from PBS pH 7.4 containing 2% BSA (US-Origin). Reconstitute with 200 µL of 50 % glycerol in deionized water. We recommend including 0.1 % sodium azide as a preservative if applicable. When reconstituted in 200 µl, the concentration of each individual single-domain antibody is 1.25 µM
kDa: -
Ext Coef: -
Shipping: Ambient temperature
Storing: Vials containing lyophilized protein can be stored at 4 °C for 6 months. We recommend reconstituting the protein with 50 % sterile glycerol including 0.1 % sodium azide as preservative if applicable. Minimize the number of freeze-thaw cycles by aliquoting the reconstituted protein. Long term storage at -80 °C for up to 6 months. Working aliquots can be stored at -20 °C for up to 4 weeks. We do not recommend storing the reconstituted protein at 4 °C.
Protocols:

This Product is not recommended to be used to detect proteins in Western Blott, sdAbs tend to recognize mainly native/folded proteins.

Look at detailed protocols and our specificity chart in our Resource Section.

 

 

References:
  1. Hunter I, Coulson B, Pettini T, et al. Balance of activity during a critical period tunes a developing network. Elife. 2024;12:RP91599. Published 2024 Jan 9. doi:10.7554/eLife.91599
  2. Rey S, Ohm H, Moschref F, Zeuschner D, Praetz M, Klämbt C. Glial-dependent clustering of voltage-gated ion channels in Drosophila precedes myelin formation. Elife. 2023;12:e85752. Published 2023 Jun 6. doi:10.7554/eLife.85752
  3. Konietzny A. Of Roadblocks and Loading Bays – Control Mechanisms of Dendritic Cargo Trafficking. PhD thesis (2020), University of Hamburg.
  4. Heck J, Parutto P, Ciuraszkiewicz A, et al. Transient Confinement of CaV2.1 Ca2+-Channel Splice Variants Shapes Synaptic Short-Term Plasticity. Neuron. 2019;103(1):66-79.e12. doi:10.1016/j.neuron.2019.04.030
Notice: To be used in vitro/ for research only. Non-toxic, non-hazardous, non-infectious.
Legal terms: By purchasing this product you agree to our general terms and conditions.