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FAQ - Nano-Traps


Questions

Answers

Q1
What is the binding capacity of the Nano-Trap (GFP-Trap/ RFP-Trap)?   A1 That depends on the beads. Agarose beads usually binds at least 5 µg GFP per 10 µl slurry, the magnetic particles around 0.25 – 0.5 µg per 10 µl slurry.
Q2
Will I be able to elute bound proteins from the GFP-Trap?   A2 For quantitative elution you can either boil the sample for 10 minutes at 95° C in SDS sample buffer 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. 
Q3
Is it possible to elute bound proteins from the GFP-Trap in their native state, e.g. for gel shift experiments or functional assays?   A3 You may try to elute with free GFP. However, please be aware that this method will not quantitatively elute your fusion protein of interest. 
Q4
How can I avoid unspecific protein interactions binding to the GFP-Trap?   A4 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. 
Q5
Will the eluted GFP binding protein cross-react with a secondary Ig specific antibody that is used to detect an antigen-specific first antibody?   A5 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. 
Q6
Is there a difference in binding when I use N-terminal vs. C-terminal GFP fusions?   A6 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) 
Q7
Can I purify GFP labeled fusion proteins directly from tissue samples, i.e. in a denaturing buffer?   A7 In principle the GFP-Trap is very stable even under harsh buffer conditions (e.g. RIPA buffer containing 0.1% SDS or 1M urea) 
Q8
Is there a size limit for GFP labeled structures that can bind to the beads of the GFP-Trap?   A8 No.
Q9
What should I do if there is residual background in my eluates?   A9 We recommend to use our blocked particles (bab-20 or bmp-20) to pre-clear your samples.
Q10
Which GFP variants does the GFP-Trap/ GFP-Booster recognize?   A10 GFP, eGFP, wtGFP, GFP S65T, TagGFP, AcGFP, eYFP, YFP, Venus, eCitrine, Citrin, CFP

 

Not recognized:

TurboGFP, all RFPs

 

Q11
Which RFP variants does the RFP-Trap/ RFP-Booster recognize?   A11 mRFP, mCherry, mOrange, mPlum, mRFPruby, mKate2, tdTomato, Strawberry

 

Not recognized:

DsRed (very weak), TagRFP, all GFPs

 

Q12
What are the biophysical parameters of GFP-Trap?   A12

Molecular weight: 14.2 kDa

Theoretical pI: 9.2

Extinction coefficient (@ 280nm): 27,000

Q13
What are the biophysical parameters of RFP-Trap?   A13

Molecular weight: 15.1 kDa

Theoretical pI: 9.2

Extinction coefficient (@ 280nm): 29,900

Q13
I’ve done a pull-down experiment on cell lysate using GFP-Trap Agarose. When using the anti-GFP antibody GMA3H9, I detect 2 different bands via Western Blot. Is this to be expected?   A13

Yes, it is common to see a second degraded form of GFP or GFP-fusion protein.

Q14
Which matrices are available for GFP-Trap?   A14

The GFP-Trap is available coupled to agarose beads, magnetic particles or magnetic agarose beads. All matrices are suitable for:

• Pulldowns/ Immunoprecipitations

• Co-IP

• Mass spectrometry

• Enzyme activity measurements

• ChIP analysis

 

Matrix properties: 

agarose beads magnetic particles magnetic agarose beads
Matrix agarose silica (non porous) agarose
Color white brown black
Particle size 90 µm 0,5-1 µm 40 µm
Binding capacity 4 µg/10 µl 0,25-0,5 µg/10 µl 4 µg/10 µl
Magnetic no yes yes
Centrifuged up to  3000 g 3000 g 3000 g

Please choose the matrix depending on your preferences.