| Q: |
Which GFP variants does the GFP-Trap® recognize? |
| A: |
GFP, eGFP, eYFP, Venus have been tested and bind equally well. |
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| Q: |
What is the binding capacity of the GFP-Trap®? |
| A: |
That depends on the beads. GFP-Trap®_A usually binds 2-3 µg GFP per 10 µl slurry, GFP-Trap®_M around 0.25 – 0.5 µg per 10 µl slurry. |
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| Q: |
Will I be able to elute bound proteins from the GFP-Trap®? |
| A: |
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. |
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| Q: |
Is it possible to elute bound proteins from the GFP-Trap® in their native state, e.g. for gel shift experiments or functional assays? |
| A: |
You may try to elute with free GFP. However, please be aware that this method will not quantitatively elute your fusion protein of interest. |
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| Q: |
How can I avoid unspecific protein interactions binding to the GFP-Trap®? |
| A: |
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. |
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| Q: |
Will the eluted GFP binding protein cross-react with a secondary Ig specific antibody that is used to detect an antigen-specific first antibody? |
| A: |
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. |
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| Q: |
Is there a difference in binding when I use N-terminal vs. C-terminal GFP fusions? |
| A: |
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) |
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| Q: |
Can I purify GFP labeled fusion proteins directly from tissue samples, i.e. in a denaturing buffer? |
| A: |
In principle the GFP-Trap® is very stable even under harsh buffer conditions (e.g. RIPA buffer containing 0.1% SDS or 1M urea) |
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| Q: |
Is there a size limit for GFP labeled structures that can bind to the beads of the GFP-Trap®? |
| A: |
No. |
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| Q: |
What should I do if there is residual background in my eluates? |
| A: |
We recommend to use our blocked particles (bab-20 or bmp-20) to preclear your samples. |
