FAQ on Nano-Traps

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

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).

Is it possible to elute bound proteins from GFP-Trap® with free GFP?

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

Does the GFP-Trap bind TurboGFP?

No, the GFP-Trap doesn't bind TurboGFP. TurboGFP is a green fluorescent protein derived from CopGFP of the copepod Pontellina plumata whereas GFP has been originally isolated from jellyfish Aequorea Victoria. Turbo-GFP shares only ~20 % sequence identity with the commonly used GFP variants.

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).

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.

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

GFP-Trap® Agarose and GFP-Trap® Magnetic Agarose usually bind around 8 µg GFP per 10 µL slurry.

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

Molecular weight: 13,9 kDa; Extinction coefficient: 27055 M-1 cm-1

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

Molecular weight: 14,9 kDa; Extinction coefficient: 30035 M-1 cm-1

Does the Myc-Trap bind endogenous c-Myc protein?

We could not detect binding of endogenous c-Myc protein to the Myc-Trap. Some epitope residues that have shown to be crucial for binding to the Myc-Trap are buried in the three-dimensional structure of the c-Myc protein. Hence, under native conditions, c-Myc protein is not a suitable binding partner for the Myc-Trap.

How can I gently elute native Myc-tagged protein from the Myc-Trap?

You can elute your Myc-fusion competitively with 1x or 2x Myc-peptide. Alternatively, you can use 8 M Urea or 0.2 M glycine pH 2.5 at room temperature.

Should I use 1x Myc- or 2x Myc-peptide for elution of the Myc-Trap?

It is likely that the Myc-Trap binds several motifs of a double Myc-tag. Hence, elution of a Myc-tagged fusion protein is more efficient with the 2x Myc-peptide. In addition, the term "Myc-tag" can refer to a single or double Myc-tag. To be on the safe side, we generally recommend to elute with the 2x Myc-peptide.

Should I use an N-terminal or C-terminal Spot-fusions? Can I also insert the Spot-Tag in the middle of my protein?

Both, an N- or C-terminal fusion work well.
The use of the Spot-Tag for internal protein tagging has to be tested case by case. The Spot-Tag peptide has to exist in a linear form and be accessible without steric hindrance from other parts of the protein of interest. An internal Spot-Tag is only likely to be recognized by the Spot-Tag Nanobody if inserted into a sufficiently large and unstructured loop, an inherently unstructured domain or a lengthy domain linker.

How can I elute bound Spot-tagged protein from the Spot-Trap in its native state?

You can elute your Spot-fusion competitively with Spot peptide. Alternatively, you can use 10 mM NaOH pH 12 (adjust pH immediately after elution).

How can I detect my Spot-fusion in Western blot application?

You can use ChromoTek's Spot-Label to detect your Spot-tagged fusion protein. Alternatively, you can use ChromoTek's Spot-Binding Protein (Spot VHH, product code: etx-250) followed by incubation with a conventional anti-Llama or anti-His6 secondary antibody.

How long should I incubate my Spot-fusion sample with Spot-Label for Western blot and IF applications?

Optimal results are achieved through overnight incubation.

Should I use an N-terminal or C-terminal fusion tag?

Both the N-terminal or C-terminal fusion tag work well with the traps.

How can I avoid unspecific protein interactions binding to the trap?

For preclearing of your sample we recommend to use our binding controls (bab-20 or bmab-20).

Please find more information in our Troubleshooting guide

How can I elute bound proteins from a trap in their native state?

You can elute your fusion protein of interest with 0.2 M glycine pH 2.5 at room temperature. Pipette the beads up and down for 60-120 seconds and repeat this step. Ensure to neutralize your supernatant immediately afterwards by adding 1 M Tris base pH 10.4.

How many mammalian cells are required for an immunoprecipitation reaction?

For one immunoprecipitation reaction, we recommend using ~10^6 - 10^7 mammalian cells. The yield is also dependent on the expression level of your protein of interest and the interaction partners.

How much cell extract should I use for an immunoprecipitation reaction?

For cell types other than mammalian cells, we recommend using 0.5 - 1.0 mg of cell extract.

Do I need to elute bound proteins from the beads for mass spectrometry analysis?

No, you can directly conduct an on-bead digestion after immunoprecipitation. This procedure allows faster and more efficient sample preparation and a potential higher yield.
Please find more information here:

Preomics kit

Protocol on-bead digestion

Do I need to elute my protein of interest from the beads for enzymatic assays?

No, you can directly perform your enzymatic assay on the beads if the active center is not blocked.

Please find more information in our application note "enzymatic activity assay"

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)*
Myc-Trap (with 2x Myc peptide): 0.5 nM, nanomolar (10-9)
Spot-Trap:  6 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).

What is the amount of trap slurry I need for one immunoprecipitation reaction?

25 µL slurry are sufficient for one pull-down reaction as the affinity of the traps is very high.

FAQ on Antibodies: Nanobodies / VHHs

Are the Nanobodies monoclonal or polyclonal?

The Nanobodies are recombinant/monoclonal.

What are the biophysical parameters of the Nanobodies?

NameMW (kDa)Molar Extinction Coefficient
(M-1 cm-1)
Theoretical pI
GFP VHH13.927.0559.4
GST VHH14.828.5458.2
Halo VHH14.823.0459.4
Histon VHH14.425.56510.2
MBP VHH14.127.05510.0
Mdm4/Hdmx VHH13.614.56510.1
MK2 VHH14.432.5559.7

mNeonGreen VHH

14.321.6808.0
Myc VHH13.523.0459.6
p53 C-term VHH14.834.1709.2
p53 N-term VHH13.020.0659.7
PARP1 VHH14.417.6709.2
RFP VHH14.930.0359.3
SNAP/CLIP-tag VHH13.124.0759.8
Spot VHH (bivalent)30.346.3405.3
TurboGFP VHH13.618.5759.5
Vimentin VHH26.748.15010.0

Do the Nanobodies have any tags?

Yes, the Nanobodies have a C-terminal His6-tag.

Does the GFP VHH bind to protein A or protein G?

The GFP VHH binds to protein A, but not to protein G.

FAQ on Antibodies: IgGs

Does the Myc-tag antibody [9E1] bind endogenous c-Myc protein?

We could not detect binding of the Myc-tag antibody [9E1] to endogenous c-Myc protein. Some epitope residues that have shown to be crucial for binding to the Myc-tag antibody are buried in the three-dimensional structure of the c-Myc protein. Hence, under native conditions, c-Myc protein is not a suitable binding partner for the Myc-tag antibody [9E1].

Can I use the Myc-tag antibody [9E1] for immunoprecipitation?

Yes, you can use the Myc-tag antibody [9E1] for immunoprecipitation and immobilize it on beads through protein A or G.

Can I use the HA-tag antibody [7C9] for immunoprecipitation?

Yes, you can use the HA antibody [7C9] for immunoprecipitation and immobilize it on beads through protein A or G.

FAQ on Nano-Boosters and Nano-Labels

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-Labels applicable for live-cell imaging?

Nano-Labels are small proteins and therefore don’t penetrate through non-permeabilized cell membranes. If you need to deliver 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-Labels.

(Valid for Histone-Label and Vimentin-Label)

Are Nano-Booster and Nano-Labels 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 .

Do Nano-Boosters work on (methanol-) fixed samples?

Yes. Nano-Booster stainings perform equally well after fixation with most common reagents: paraformaldehyde, glutaraldehyde, methanol (Kaplan & Ewers, 2015; Ries et al., 2012).

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.

FAQ on Chromobodies

Are Chromobodies constitutively expressed?

Yes, Chromobody expression is regulated by immediate early promotor CMV. This promotor allows constitutive Chromobody expression.

Do Chromobodies only work in live cells?

Yes, the Chromobody plasmid is only expressed in live cells. Cells should be transfected with the Chromobody plasmid at least overnight to observe the Chromobody location signal. Alternatively, cells can be fixed prior to imaging.
Note: We don't recommend fixation of cells for the Histone-Chromobody.

When should I image my cells after transfection with the Chromobody plasmid?

The Chromobody signal is maintained up to 3 days in the cell. However, this also depends strongly on the cell type.
We recommend to image the cells 16-24 hours after transfection.

Do the Chromobodies diffuse through the cell membrane into growth medium?

No, Chromobodies are small proteins being expressed in the cytosol. They are not secreted into the medium and remain in the cell as long as the cell maintains its plasma membrane integrity.

Are Chromobodies fluorogenic or do they only emit fluorescence when bound to a target?

Chromobodies are chimeric proteins consisting of a VHH fused to a fluorescent protein. They maintain their fluorescence regardless of whether they are bound to a target or not.

Can I amplify the Chromobody plasmid in bacteria?

Yes, the Chromobody plasmids can be propagated in E.coli by standard techniques.

FAQ on Accessories

Can I recover the beads from the spin column?

Yes, you can transfer the beads from the spin columns. Add 500 µL buffer to the spin column, pipette up and down and transfer the buffer-bead suspension to a fresh tube. Sediment the beads for 2 min at 2.500x g and room temperature and remove the buffer.