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Which beads should I use for my immunoprecipitation?

Posted by Dr. Astrid Sitte on Sep 16, 2020 9:42:57 AM

Directions_Nano-Trap

ChromoTek's Nano-Traps are optimized for the immunoprecipitation (IP) of proteins and their interacting factors. Nano-Traps comprise of a Nanobody/ VHH conjugated to beads. For 3 Nano-Traps, e.g. GFP-Trap®RFP-Trap®, and Spot-Trap®, we offer different types of beads:

  • Agarose beads
  • Magnetic Agarose beads
  • Dynabeads™

ChromoTek_GFP-Trap_Agarose

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

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

  • Agarose beads for very low background and high binding capacity IP
  • Magnetic Agarose beads for magnetic separation and high binding capacity IP
  • Dynabeads for IP of very large proteins/complexes

 

Agarose

Magnetic Agarose

Dynabeads

Low background*

+++

++

++

Binding capacity

+++

+++

+

Size of 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 have to be tested.

In detail, all beads have very low background binding due to the highly specific binding of the Nano-Traps. In comparison, Agarose beads have the lowest background. The background of Magnetic Agarose beads and Dynabeads is at slightly higher. Hence, Agarose is recommended if a very low background is needed.

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

The size and shape of your protein of interest (POI) may require the use of the non-porous Dynabeads, because larger proteins or protein complexes can’t access the Nanobodies/ VHHs 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.

 

Beads

 

Agarose

Magnetic Agarose

Dynabeads

Matrix

Agarose
(4% cross-linked)

Magnetic agarose
(6% cross linked)

Dynabeads M-270

Bead form

Porous

Porous; sold iron core

Solid

Ligand

Nanobody/ VHH

Nanobody/ VHH

Nanobody/ VHH

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

Very high

Very high

Medium

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

GFP-Trap Agarose
GFP-Trap Magnetic Agarose
GFP-Trap Dynabeads

RFP-Trap Agarose
RFP-Trap Magnetic Agarose
RFP-Trap Dynabeads

Spot-Trap Agarose
Spot-Trap Magnetic Agarose
Spot-Trap Dynabeads

Please contact support@chromotek.com for more details.

ChromoTek, GFP-Trap, RFP-Trap, and Spot-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, Immunoprecipitation, RFP-Trap, GFP Immunoprecipitation, GFP Nanobody, Spot-Trap, Dynabeads

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