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).
You may try to elute with free GFP. However, please be aware that this method will not quantitatively elute your fusion protein of interest.
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.
In principle the GFP-Trap® is very stable even under harsh buffer conditions (e.g. RIPA buffer containing 0.1% SDS or 1M urea).
Molecular weight: 13,9 kDa; Extinction coefficient: 27055 M-1 cm-1
Molecular weight: 14,9 kDa; Extinction coefficient: 30035 M-1 cm-1
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.
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.
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.
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.
You can elute your Spot-fusion competitively with Spot peptide. Alternatively, you can use 10 mM NaOH pH 12 (adjust pH immediately after elution).
Optimal results are achieved through overnight incubation.
Both the N-terminal or C-terminal fusion tag work well with the traps.
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.
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.
For cell types other than mammalian cells, we recommend using 0.5 - 1.0 mg of cell extract.
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"
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).
25 µL slurry are sufficient for one pull-down reaction as the affinity of the traps is very high.
|Name||MW (kDa)||Molar Extinction Coefficient|
|p53 C-term VHH||14.8||34.170||9.2|
|p53 N-term VHH||13.0||20.065||9.7|
|Spot VHH (bivalent)||30.3||46.340||5.3|
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].
Yes, you can use the Myc-tag antibody [9E1] for immunoprecipitation and immobilize it on beads through protein A or G.
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
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.
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.
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.
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.
Yes. Nano-Booster stainings perform equally well after fixation with most common reagents: paraformaldehyde, glutaraldehyde, methanol (Kaplan & Ewers, 2015; Ries et al., 2012).
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.
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.
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.
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.
Yes, Chromobody expression is regulated by immediate early promotor CMV. This promotor allows constitutive Chromobody expression.
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.
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.
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.
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.
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.