FAQs

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Which GFP variants does the GFP-Trap® recognize?

• eGFP, wtGFP, GFP S65T
• TagGFP
• eYFP, YFP, Venus, Citrin
• CFP

Not recognized:

• TurboGFP
• all RFPs

Which RFP variants does the RFP-Trap® recognize?

• mRFP
• mCherry
• mOrange
• mPlum
• mRuby
• mKate2

Not recognized:

• DsRed
• mRFPruby
• TagRFP
• all GFPs

What is the binding capacity of the Nano-Trap (GFP-Trap®/ RFP-Trap®)?

That depends on the beads. Agarose beads usually bind 2-3 µg GFP per 10 µl slurry, the magnetic particles around 0.25 - 0.5 µg per 10 µl 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 the 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 0.4 ml. In addition we recommend to test various salt concentrations in the wash buffer (e.g. 150 mM - 500 mM NaCl) to remove unspecifically bound hydrophilic proteins.

Will the eluted GFP binding protein cross-react with a secondary Ig specific antibody that is used to detect an antigen-specific first 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 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 residual background in my eluates?

We recommend to use our blocked particles (bab-20 or bmp-20) to preclear your samples.

What is the difference between a Nanobody and your GFP-Trap®?

Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies, developed by Belgian company Ablynx.
While the GFP-Trap^® is also derived from heavy-chain antibodies, we do not call it GFP nanobody, since nanobodies are therapeutic proteins. GFP-Trap^® in contrast is your premier tool for biochemical studies of your GFP fusion proteins.