ChromoTek FAQ
1. What are the types of GFP derivatives that GFP-Trap® recognizes?
(1) eGFP, wtGFP, GFP S65T
(2) TagGFP
(3) eYFP, YFP, Venus, Citrin
(4) CFP
Unrecognized: TurboGFP, all RFPs
2. What are the types of RFP derivatives that RFP-Trap® recognizes?
(1) mRFP
(2) mCherry
(3) mOrange
(4) mPlum
(5) mRuby
(6) mKate2
Not recognized: DsRed, mRFPruby, TagRFP, all GFPs
3. What is the binding capacity of Nano-Trap® (GFP-Trap® / RFP-Trap®)?
The ability to bind Nano-Trap® depends on the beads. For each 10 μL of the slurry, the agarose beads can bind 2 to 3 μg of GFP, and the magnetic particles can bind 0.25- to 0.5 μg of GFP.
4. Can I elute binding proteins from GFP-Trap?
For quantitative elution, the sample can be boiled in SDS loading buffer at 95 ° C for 10 min (see protocol) or in 0.2 M glycine (pH 2.5) for 0.5 to 2 min, followed by 0.1 volume of 1 M Tris base. neutralize.
5. Is it possible to elute a natural binding protein from GFP-Trap, for example, in a gel migration assay or a functional assay?
You can try to elute free GFP. However, please note that this method does not quantitatively elute the target fusion protein.
6. How can I avoid the combination of non-specific proteins interacting with GFP-Trap?
The key operating step is to dilute the detergent concentration in the incubation solution. We recommend that the detergent be at a final concentration of 0.1% (eg NP-40 or TX-100) and the final volume is not less than 0.4 mL. In addition, we propose to determine the salt concentration of various wash buffers ranging from 150 mM to 500 mM to remove non-specifically bound hydrophilic proteins.
7. Is there a difference between the N-terminal and C-terminal GFP fusion proteins during the binding process?
GFP-Trap® has a slightly higher affinity for the C-terminal GFP fusion protein. To eliminate this difference, the incubation time can be lengthened (1-2h instead of 15-30min).
8. Can I directly purify GFP-tagged fusion proteins in tissue samples (ie, denaturing buffer)?
In principle, GFP-Trap® is very stable even under harsh buffer conditions (eg RIPA buffer with 0.1% SDS or 1 M urea).
9. Is there a size limit for the GFP structure of the GFP-Trap® beads that can be combined?
no.
10. What if there is a remaining background in the eluent?
Samples are pre-treated with Chromotek's blocked particles (bab-20 or bmp-20).
For more questions, please contact ChromoTek China General Agent Shanghai Murray Bio, Hotline: 4006-400-850
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