GFP-Trap Dynabeads

Description
GFP-Trap® Dynabeads for immunoprecipitation (IP) of GFP-tagged proteins. GFP-Trap® Dynabeads is highly recommended, when very large proteins/complexes are investigated, and magnetic separation is needed for IP.
It consists of a GFP VHH/ Nanobody coupled to DynabeadsTM

Specificity
EGFP, GFP, CFP, YFP, BFP and many more derivatives, see: Fluorescent protein specificity table (PDF)

Applications
Immunoprecipitation/ Co-immunoprecipitation
Mass spectrometry
On-bead enzyme assays

Product Size Code Price Buy
Product GFP-Trap Dynabeads Size 250 µL (10 reactions) Code gtd-10 Price $ 335
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Product GFP-Trap Dynabeads Size 500 µL (20 reactions) Code gtd-20 Price $ 550
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Product GFP-Trap Dynabeads Size 2.5 mL (100 reactions) Code gtd-100 Price $ 2475
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Product GFP-Trap Dynabeads Size 5 mL (200 reactions) Code gtd-200 Price Please inquire
Product GFP-Trap Dynabeads Size 10 mL (400 reactions) Code gtd-400 Price Please inquire
Product GFP-Trap Dynabeads Kit Size 500 µL (20 reactions) Code gtdk-20 Price $ 687.5
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Product GFP VHH, recombinant binding protein Size 250 µL Code gt-250 Price $ 275
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Affinity
Dissociation constant KD of 1 pM

Wash buffer compatibility
10 mM DTT, 8 M Urea, 2 M NaCl, 2 % Nonidet P40 Substitute, 0.2 % SDS, 1 % Triton X-100

Specificity (selection)
- AcGFP, Clover, eGFP, Emerald, GFP, GFP5, GFP Envy, GFP S65T, mGFP, mPhluorin, PA-GFP, Superfolder GFP, TagGFP, TagGFP2, monomeric eGFP A206K
- CFP
- YFP, Citrine, eCitrine, eYFP, Venus, Ypet
- BFP
For complete list, please click here: Fluorescent protein specificity table

Binding capacity
10 µL slurry bind about 1 µg of recombinant GFP

Coupled Nanobody/ VHH
Monoclonal anti-Green Fluorescent Protein (GFP) single domain antibody (sdAb) fragment, recombinantly expressed

Bead properties
Bead size: ~ 2.8 µm
Magnetic, high throughput-compatible
Storage buffer: 1x PBS, Preservative: 0.09 % sodium azide

Elution
- SDS sample buffer
- 0.2 M glycine pH 2.5
Instead of elution, we recommend on-bead assays like on-bead digestion for MS analysis.

Compatibility with mass spectrometry
The GFP-Trap is optimized for on-bead digestion. Complete tryptic digest results in 4-5 peptides.

Storage instructions
Shipped at ambient temperature. Upon receipt store at 4°C. Stable for 1 year.

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).

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, GFP-Trap Dynabeads binds around 1 µg 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

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) when you use GFP-Trap Agarose or Magnetic Agarose for the IP.

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 other type of cells 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.

Specifications of GFP-Trap Agarose, Magnetic Agarose, and Dynabeads

 

GFP-Trap

 

Agarose

Magnetic Agarose

Dynabeads

Matrix

Agarose (4% cross-linked)

Magnetic agarose (6% cross linked)

Dynabeads M-270

Bead form

porous

Porous; sold iron core

solid

Ligand

GFP VHH

GFP VHH

GFP VHH

GFP-tagged protein size*

Small to large size

Small to large size

Small to very large size; no size limitation

Color

White

Black

Brown

Medium particle size

90 µm

40 µm

2.8 µm

Binding capacity

12 µg/ 10 µL

8 µg/ 10 µL

1 μg/ 10 μL

Background

Very low

Low

Low

Magnetic separation & automation

no

yes

yes

May be centrifuged up to

2,500 x g

800 x g

8,000 x g

* Does depend on protein size and shape, protein multimers, complexes and interaction partners

 

Should I preclear my sample when using GFP-Trap Dynabeads?

The Dyanbeads matrix is inert and shouldn’t bind background proteins. Hence, unconjugated Dynabeads shouldn’t be used for preclearing. To investigate unspecific binding to GFP-Trap Dynabeads, we recommend to perform the IP with mock cell lysate without GFP-fusion or with GFP only.

Benefits

  • IP and Co-IP of GFP-tagged proteins without size limitation
  • Magnetic separation with easy and efficient washing of GFP-Trap Dynabeads
  • Automation and high throughput applications
  • No heavy & light antibody chains, short incubation (5-30 min)
  • Extraordinary binding, also under harsh conditions
  • Very high affinity (KD=1 pM) to bind even low abundant proteins

Why protein size matters in immunoprecipitation

The immunoprecipitation of very large proteins like oligomers or complexes and the Co-IP of bulky/multiple binding partners may be challenging when using porous beads. The pores of agarose or magnetic agarose beads have a certain size and hence may exclude proteins, multimers, or complexes to diffuse into the pores and to interact with the Nanobody ligand; next to size also the protein’s shape may limit diffusion. Therefore, binding can just occur on the outer surface of the agarose or magnetic agarose beads, which results in a poor immunoprecipitation performance. 
Dynabeads are non-porous, solid particles with ligands coupled on their surface. Hence all size GFP-tagged proteins including very large GFP-tagged proteins, oligomers and complexes including bulky binding partners bind to the GFP Nanobody of GFP-Trap Dyanbeads and are effectively immunoprecipitated.

GFP-Trap Dynabeads Kit

The GFP-Trap Dynabeads is also available in a kit, including:

  • GFP-Trap Dynabeads
  • lysis*, wash and elution buffers

*The lysis buffer provided is suitable for mammalian cells. For other cell types use another suitable lysis buffer.

Which GFP-Trap should I use?

  • GFP-Trap Agarose, when lowest background and high binding capacity IP is needed. 
  • GFP-Trap Magnetic Agarose, when magnetic separation and high binding capacity IP is needed.
  • GFP-Trap Dynabeads, when very large proteins/complexes are investigated, and magnetic separation is needed for IP.
  GFP-Trap Agarose  GFP-Trap Magnetic Agarose  GFP-Trap Dynabeads 
Low background  +++  ++  ++
Binding capacity  +++  +++  +
Size GFP-tagged protein*   Small to large  Small to large  Small to very large
Bead separation  centrifugation  magnetic  magnetic

* Does depend on protein size and shape, protein multimers, complexes and interaction partners

Testimonials

 “We compared the GFP-Trap Dynabeads with the MA beads and the Dynabeads are far superior not only for the amount of bait pull down, but importantly also of its interaction partner."
Lecturer in Cell Biology, University of Cambridge
 

I’ve tried GFP-Trap Dynabeads sample that I received with my last order and it’s working perfectly!
PhD student, Institut Curie, France


The new optimised GFP-Trap Dynabeads are indeed better than the GFP-Trap beads that we were using so far, for proteins larger than 200 kDa.
Postdoctoral fellow, University of Groningen

 

On my protein, which forms pentamers and also complexes with many other proteins, the GFP-Trap Dynabeads  worked at least as well as the agarose. Moreover, the manipulation with magnetic beads is much more easier.
Researcher, Institute of Hematology and Blood, Prague

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Only for research applications, not for diagnostic or therapeutic use!