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What GFP-Trap should I use for my immunoprecipitation?

Posted by Dr. Astrid Sitte on Jan 20, 2020 4:42:38 PM

Directions_GFP-Trap-1

ChromoTek GFP-Trap® is optimized for the immunoprecipitation of GFP-tagged proteins and their interacting factors.

The GFP-Trap consists of the ChromoTek anti-GFP Nanobody/ VHH that is coupled to 3 different types of beads:

  • Agarose beads
  • Magnetic Agarose beads
  • Dynabeads™

Based on the properties of the different beads (see table), we recommend:

ChromoTek_GFP-Trap_AgaroseImmunoprecipitation with GFP-Trap.
I: Input, FT: Flow-Through, B: Bound

GFP-Trap

Agarose

Magnetic Agarose

Dynabeads

Low background*

+++

++

++

Binding capacity

+++

+++

+

Size GFP-tagged protein*

Small to large

Small to large

Small to very large

Bead separation

centrifugation

magnetic

magnetic

*Please note that these are general guidelines. For a specific protein or complex, the optimal experimental conditions/ GFP-Trap have to be tested.

In detail, all types of GFP-Trap have very low background binding due to the highly specific binding of GFP (fusion proteins). In comparison, GFP-Trap Agarose has the lowest background. The background of GFP-Trap Magnetic Agarose and GFP-Trap Dynabeads is at slightly elevated levels. Hence, GFP-Trap Agarose is recommended if a very low background is needed.

For automated immunoprecipitation or when magnetic handling is preferred in general, GFP-Trap Magnetic Agarose or GFP-Trap Dynabeads shall be used. However, GFP-Trap Magnetic Agarose is recommended if a higher binding capacity is required.

The size and shape of the actual GFP-fusion protein, multimer or complex may require the use of the non-porous GFP-Trap Dynabeads, because larger GFP-fusion proteins can’t access the GFP VHH ligands inside the pores of agarose and magnetic agarose beads. Note that smaller proteins may form multimers or may be part of larger complexes and hence have larger sizes than expected.


 

GFP-Trap

 

Agarose

Magnetic Agarose

Dynabeads

Matrix

Agarose
(4% cross-linked)

Magnetic agarose
(6% cross linked)

Dynabeads M-270

Bead form

Porous

Porous; solid iron core

Solid

Ligand

GFP VHH

GFP VHH

GFP VHH

Affinity to GFP

1 pM

1 pM

1 pM

GFP-tagged protein size*

Small to large size

Small to large size

Small to very large size; all sizes

Color

White

Black

Brown

Medium particle size

~90 µm

~40 µm

2.8 µm

Binding capacity

12 µg/ 10 µL

8 µg/ 10 µL

1 μg/ 10 μL

Background

Very low

Low

Low

Magnetic separation & automation

No

Yes

Yes

May be centrifuged up to

2,500 x g

800 x g

8,000 x g

* Does depend on protein size and shape, protein multimers, complexes and interaction partners

Please contact support@chromotek.com for more details.

 

ChromoTek and GFP-Trap are registered trademarks of ChromoTek GmbH, Nanobody is a registered trademark of Ablynx, a Sanofi company, and Dynabeads™ is a trademark of Life Technologies AS, a part of Thermo Fisher Scientific Inc.

Topics: GFP-Trap, GFP Immunoprecipitation, GFP Nanobody, Dynabeads

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