RFP-Booster

Description:
RFP-Booster consists of an RFP-Binding protein (also termed RFP nanobody) coupled to fluorescent dyes for IF/microscopy:

Specificity:
RFP-Booster specifically binds to most common RFP derivatives:

  • mRFP, mCherry, mRFPruby, mPlum, DsRed
     

Application:
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 1/1600

Form: Purified recombinant protein in PBS

Size: 10μl, 100μl

Protein concentration:
0.5 – 1 mg/ml (conjugates)

Preservative: 0.09% sodium azide

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

  

RFP-Booster specifically labels RFP-fusion proteins. HeLa cells expressing mRFP-PCNA, fixed and permeablilized. Specific staining of mid replication foci with RFP-Booster_Atto594.

Enhancement of RFP signal with RFP-Booster_Atto594. Comparison of signal intensity of a cell line stably expressing a nuclear RFP-fusion protein before and after RFP-Booster treatment.

Ordering information

Prices in EUR

ProductCodeVolumePriceBuy
RFP-Booster_Atto594rba594-1010µl75 EUR Cart
RFP-Booster_Atto594rba594-100100µl270 EUR Cart
RFP-Booster_Atto647Nrba647n-1010µl85 EUR Cart
RFP-Booster_Atto647Nrba647n-100100µl295 EUR Cart
RFP-Binding protein,
purified protein for conjugation, 1 g/l
rt-250250µl215 EURCart
RFP-Binding protein, custom labelingCustomPlease inquire

Prices in USD

ProductCodeVolumePriceBuy
RFP-Booster_Atto594rba594-1010µl100 USD Cart
RFP-Booster_Atto594rba594-100100µl365 USD Cart
RFP-Booster_Atto647Nrba647n-1010µl110 USD Cart
RFP-Booster_Atto647Nrba647n-100100µl395 USD Cart
RFP-Binding protein,
purified protein for conjugation, 1 g/l
rt-250250µl275 USDCart
RFP-Binding protein, custom labelingCustomPlease inquire
Protocol, SDS, etc.
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FAQs

Do Boosters work on methanol-fixed samples?
Yes. Booster stainings perform equally well after fixation with most common reagents: paraformaldehyde, glutaraldehyde, methanol (Kaplan & Ewers, 2015; Ries et al., 2012).

Are Boosters applicable for live-cell imaging?
Yes, if the GFP/RFP-tag is on the cell surface.
Being a 15 kDa protein, Booster does not penetrate through unpermeabilized cell membranes. Hence, if your GFP is intracellular, you have to fix and permeabilize the cells. Alternatively, it is possible to microinject or electroporate the Booster protein into the cells for live-cell imaging, but these approaches are rarely used.

What is the protocol for live-cell Booster staining of the extracellular GFP/RFP-tag?

Incubate the cells with 1:25 Booster 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 penetrate though the cell membranes of live cells?

No. Nano-Boosters are 15 kDa in size and they do not penetrate through unpermeabilized cell membranes. With different protein transduction methods and reagents it is possible to introduce some small amounts of Nano-Boosters into live cells, but the most efficient way is to microinject the Boosters.

Can I do a simultaneous co-staining with GFP and RFP-Boosters?
Yes, you can combine the Boosters. For example, if you normally use the Boosters in 1:200 dilution, for a co-staining you should add both 1µL of gba488, as well as 1µL rba594 to 200µL of blocking solution.

Which Booster conjugates are recommended for super-resolution microscopy?
Nano-Boosters are highly suitable for super-resolution microscopy. Due to their small size (2-3nm), they minimize the linkage error and provide a more precise and dense staining, than conventional antibodies. The choice of a Booster conjugate depends on your microscope setup and lasers. We recommend:
STED: ATTO647N, Abberior STAR 635P
STORM: ATTO488/594/647N, or custom conjugation to Alexa647
SIM: ATTO488/594

Is it possible to conjugate Boosters to other dyes?
Yes. You can label our unconjugated GFP-Binding Protein (15 kDa; code: gt-250) with an NHS-dye of choice following the instructions of the dye manufacturer.
Alternatively, you can make use of our service for custom labeling of the Booster. Please send your inquiries to info(at)chromotek(dot)com.

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 www.ncbi.nlm.nih.gov/pubmed/25099357 and Platonova et al., ACS Chem Biol 2015 www.ncbi.nlm.nih.gov/pubmed/25806422

Are Boosters suitable for doing IF in yeast?
Yes, immunostaining of yeast with Nano-Boosters is simpler than with conventional antibodies, since due to their small size Nano-Boosters can penetrate the yeast cell wall. An optimized protocol for yeast staining with Nano-Boosters (GFP nanobody) is detailed in Kaplan & Ewers, Nat Protoc. 2015: www.ncbi.nlm.nih.gov/pubmed/26068895