Questions on Nano-Traps:

What is the binding capacity of the GFP-Trap®?

That depends on the beads. GFP-Trap®_A and GFP-Trap®_MA usually bind around 8µg GFP per 10µl slurry, GFP-Trap®_M around 0.25 – 0.5µg per 10µl slurry.

Will I be able to elute bound proteins from the GFP-Trap®?

For quantitative elution you can either boil the sample for 10 minutes at 95° C in SDS sample buffer (see protocol) or incubate for 0.5 to 2 minutes in 0.2M glycine pH 2.5, followed by neutralization with 1/10 vol 1M Tris base.

Is it possible to elute bound proteins from GFP-Trap® in their native state, e.g. for gel shift experiments or functional assays?

You may try to elute with free GFP. However, please be aware that this method will not quantitatively elute your fusion protein of interest.

How can I avoid unspecific protein interactions binding to the GFP-Trap®?

The critical step is to dilute the concentration of the detergent in the incubation buffer. We recommend a final concentration of 0.1% of the detergent (e.g. NP-40 or TX-100) and a final volume of at least 0.4ml. In addition we recommend to test various salt concentrations in the wash buffer (e.g. 150mM – 500mM NaCl) to remove unspecifically bound hydrophilic proteins.

Will the eluted GFP binding protein cross react with a secondary Ig specific antibody?

Since the binding protein used in the GFP-Trap® does not have any significant homology with goat, mouse, rat or human antibodies, unspecific reactions with a secondary Ig specific antibody should not occur.

Is there a difference in binding when I use the N-terminal vs. C-terminal GFP-fusions?

The GFP-Trap® has a slightly higher affinity for C-terminal GFP-fusions. You can compensate this by an elongated incubation time ( 1 - 2 h instead of 15 – 30 min)

Can I purify GFP labeled fusion proteins directly from tissue samples, i.e. in a denaturing buffer?

In principle the GFP-Trap® is very stable even under harsh buffer conditions (e.g. RIPA buffer containing 0.1% SDS or 1M urea)

Is there a size limit for GFP labeled structures that can bind to the beads of the GFP-Trap®?

No.

What should I do if there is a residual background in my eluates?

We recommend to use our binding controls (bab-20, bmab-20 or bmp-20) to preclear your samples.

Which GFP variants does the GFP-Trap® recognize?

GFP-Trap® recognizes:

  • eCFP, CFP, mCerulean
  • eGFP, wtGFP, GFP S65T, AcGFP, TagGFP, tagGFP2, sfGFP, pHluorin
  • eYFP, YFP, Venus, Citrine

Which RFP variants does the RFP-Trap® recognize?

What are the biophysical parameters of the GFP-Trap®?

Molecular weight: 14,1kDa
Extinction coefficient: 27055 M-1 cm-1

What are the biophysical parameters of the RFP-Trap®?

Molecular weight: 15,1 kDa
Extinction coefficient: 30035 M-1 cm-1

Which matrices are available for GFP-Trap®?

 

Available matrices & properties

agarose beadsmagnetic agarose beadsmagnetic particles
matrixagaroseagarosesilica (non porous)
colourwhiteblackbrown
particle size90µm40µm0,5-1µm
binding capacity12µg/10µl8µg/10µl0,25-0,5µg/10µl
magneticnoyesyes
may be centrifuged up to 3000 x g3000 x g3000 x g

What are the dissociation constants of the Nano-Traps?

Generally heavy chain antibodies do have high affinities to their antigens with dissociation constants in the low nanomolar down to the picomolar range. ChromoTek has determined the following KD values:

GFP-Trap:          1 pM, picomolar (10-12 molar)*

RFP-Trap:          5 nM, nanomolar (10-9 molar)

MBP-Trap:         4 nM, nanomolar (10-9 molar)

GST-Trap:          1 nM, nanomolar (10-9 molar)*

 

*Kinetic parameter has been measured using the switchSENSE technology using electro-switchable nanolevers to analyze molecular interactions. switchSENSE is a proprietary technology from Dynamic Biosensors (www.dynamic-biosensors.com).

Questions on Nano-Boosters:

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 (2-3 nm), 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 http://www.ncbi.nlm.nih.gov/pubmed/25099357 and Platonova et al., ACS Chem Biol 2015 http://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

Which GFP variants does the GFP-Booster recognize?

GFP-Booster recognizes:

  • eCFP, CFP, mCerulean
  • eGFP, wtGFP, GFP S65T, AcGFP, TagGFP, tagGFP2, sfGFP, pHluorin
  • eYFP, YFP, Venus, Citrine

Which RFP variants does the RFP-Booster recognize?

RFP-Booster recognizes mRFP, mCherry, mRFPruby, tdTomato, mPlum