GFP-Trap Agarose

Description

GFP-Trap® Agarose is an affinity resin for immunoprecipitation of GFP-fusion proteins.
It consists of a GFP Nanobody/ VHH coupled to agarose beads.

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

Applications
Immunoprecipitation/ Co-IP
Mass spectrometry
On-bead enzyme assays
RIP analysis

Product Size Code Price Buy
Product GFP-Trap Agarose Size 250 µL (10 reactions) Code gta-10 Price $ 275
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Product GFP-Trap Agarose Size 500 µL (20 reactions) Code gta-20 Price $ 475
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Product GFP-Trap Agarose Size 2.5 mL (100 reactions) Code gta-100 Price $ 2050
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Product GFP-Trap Agarose Size 5 mL (200 reactions) Code gta-200 Price Please inquire
Product GFP-Trap Agarose Size 10 mL (400 reactions) Code gta-400 Price Please inquire
Product GFP-Trap Agarose Kit Size 500 µL (20 reactions) Code gtak-20 Price $ 595
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Product Binding Control Agarose Beads Size 500 µL (20 reactions) Code bab-20 Price $ 75
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Product Spin Columns Size 10 units Code sct-10 Price $ 35
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Product Spin Columns Size 20 units Code sct-20 Price $ 60
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Product Spin Columns Size 50 units Code sct-50 Price $ 130
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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
1 mM DTT, 3 M Guanidinium•HCl, 8 M Urea, 2 M NaCl, 2 % Nonidet P40 Substitute, 1 % 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, eCFP, mCerulean
- YFP, Citrine, eCitrine, eYFP, Venus, Ypet
- BFP
For complete list, please click here: Fluorescent protein specificity table

Binding capacity
10 µL slurry bind about 12 µg of recombinant GFP

Coupled Nanobody/ VHH
Recombinant, monoclonal anti-Green Fluorescent Protein (GFP) single domain antibody (sdAb) fragment

Bead properties
Bead size: ~ 90 µm (cross-linked 4 % agarose beads)
Storage buffer: 20 % EtOH

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.

RRID
AB_2631357

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

Laura Trinkle-Mulcahy, Séverine Boulon, Yun Wah Lam , Roby Urcia , François-Michel Boisvert , Franck Vandermoere, Nick A. Morrice, Sam Swift, Ulrich Rothbauer, Heinrich Leonhardt, Angus Lamond. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J. Cell Biol. Vol. 183 No. 2 223–239

Zoltan Lipinszki, Peng Wang, Rhys Grant, Catherine Lindon, Nikola S. Dzhindzhev, Pier Paolo D’Avino, Marcin R. Przewloka, David M. Glover, Vincent Archambault; Affinity Purification of Protein Complexes from Drosophila Embryos in Cell Cycle Studies. Methods Mol Biol. 2014;1170:571-88. doi: 10.1007/978-1-4939-0888-2_33.

Arne H. Smits, Pascal W. T. C. Jansen, Ina Poser, Anthony A. Hyman, Michiel Vermeulen; Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics. Nucleic Acids Res 2013; 41 (1): e28. doi: 10.1093/nar/gks941

Benedetta Turriziani, Amaya Garcia-Munoz, Ruth Pilkington, Cinzia Raso, Walter Kolch, Alexander von Kriegsheim; On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics. Biology 2014, 3(2), 320-332; doi:10.3390/biology3020320

David R. Croucher, Mary Iconomou, Jordan F. Hastings, Sean P. Kennedy, Jeremy Z. R. Han, Robert F. Shearer, Jessie McKenna, Adrian Wan, Joseph Lau, Samuel Aparicio, Darren N. Saunders; Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers. Sci. Signal. 12 Jul 2016 : ra69.

Timothy D. Cummins and Gopal P. Sapkota. Characterization of protein complexes using chemical cross-linking coupled electrospray mass spectrometry. ArXiV 2016 arxiv.org/ftp/arxiv/papers/1606/1606.04247.pdf

"Jennifer N. Byrum, Shuying Zhao, Negar S. Rahman, Lori M. Gwyn,William Rodgers, and Karla K. Rodgers
An Interdomain boundary in RAG1 facilitates cooperative binding to RAG2 in formation of the V(D)J recombinase complex"

"Julian R. Avila,a,b Jin Suk Lee,a,b and Keiko U. Torii
Co-Immunoprecipitation of Membrane-Bound Receptors"

 

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.

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).

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.

Benefits

  • Effective pulldown of GFP-fusion proteins for consistent results
  • 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
  • Reliable gold standard with more than 1,500 publications

GFP-Trap Agarose Kit

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

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

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

Spin Columns

  • Easy and convenient handling
  • Rapid washing and clean elution of bound proteins
  • Simplify the pulldown of your protein
  • Spin Columns for GFP-Trap Agarose

Binding Control for Agarose Beads

  • Control for unspecific binding of proteins, DNA, etc. to beads
  • Pre-clearing of cell lysate
  • Binding Control for GFP-Trap Agarose

Testimonials

 “Your GFP-Trap® is great! I have never seen such efficient reagent for pull down."
Prof. Dr. Tomoyuki Tanaka, Wellcome Trust Centre for Gene Regulation & Expression, University of Dundee
 

We recently had excellent MS results with the GFP-Trap®.
Dr. Gwyneth Ingram, IMPS, Edinburgh


We have been pleased with the results from both of the products: GFP-Trap_A® and GFP-Trap_M®
Katharine S. Ullman, Ph.D., Assoc. Professor, Huntsman Cancer Institute, University of Utah

 

I just wanted to give you a quick update on our experiences testing your GFP-Trap®. This will be quick because our experience is that they work fantastically well!
Prof. Dr. A.I. Lamond, Wellcome Trust Center, University of Dundee

 

“We were gob smacked when we did our own IP and saw the glowing green GFP-Trap® beads...”
Prof. Dr. Laura Trinkle-Mulcahy, University of Ottawa

 

The GFP-Trap® worked very nicely to pull-down our actin binding protein...
Prof. Dr. Evelyne Friederich, University of Luxembourg

 

I am brim over with enthusiasm for your GFP-Trap®; great product and great idea; Hats off!
Dr. Monika Jedrusik-Bode, MPI for Biophysical Chemistry, Göttingen

 

"GFP-Trap® is the best beads ever!"

Dr. Kenkyo Matsuura, Department of Pharmacology and Cancer Biology, Duke University

 

“When I used antibody for immunoprecipitation, strong background was usually hard trouble. I think that GFP-Trap® is revolution.“
Takeshi Mizuno, Ph. D., Advanced Science Institute, RIKEN (Institute of Physical and Chemical Research)

 

"The results obtained with the GFP-Trap® are much more clean (…) I am thus very happy about the GFP-Trap_A® product!”
Florence Besse, PhD, Univ. Nice Sophia-Antipolis

 

Man erlebt es in der Wissenschaft ja eher selten, dass ein neues Tool in den eigenen Händen genau so gut funktioniert, wie man es sich erhofft hatte - mit der GFP-Trap_A® war das bei mir der Fall! Das hat mich wirklich überzeugt!
Friederike Althoff, Institut of Zoology, University of Zürich

 

The beads GFP-Trap®_M are fantastic!
Francesca Sicilia, Dipartimento di Biologia Vegetale, Universitá  di Roma "La Sapienza"

 

"Your GFP-Trap® is such a powerful tool for bio-sensing applications! Stable, specific, sensitive, soluble...it has all the ideal features of a capturing molecule. And it comes at a very competitive price."
Dr. Eduardo Della Pia, Nano Science Center, University of Copenhagen

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