RFP-Trap Dynabeads

Description
RFP-Trap® Dynabeads for immunoprecipitation (IP) of RFP-tagged proteins. RFP-Trap Dynabeads is highly recommended, when very large proteins/complexes are investigated, and magnetic separation is needed for IP.
It consists of a RFP VHH/ Nanobody coupled to DynabeadsTM

Specificity
mCherry, mRFP, mPlum, tagRFP, mKate2 and more RFP derivatives; see: Fluorescent protein specificity table (PDF)

Applications
Immunoprecipitation/ Co-immunoprecipitation
Mass spectrometry
On-bead enzyme assays

Product Size Code Price Buy
Product RFP-Trap Dynabeads Size 250 µL (10 reactions) Code rtd-10 Price $ 335
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Product RFP-Trap Dynabeads Size 500 µL (20 reactions) Code rtd-20 Price $ 550
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Product RFP-Trap Dynabeads Size 2.5 mL (100 reactions) Code rtd-100 Price $ 2475
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Product RFP-Trap Dynabeads Size 5 mL (200 reactions) Code rtd-200 Price Please inquire
Product RFP-Trap Dynabeads Size 10 mL (400 reactions) Code rtd-400 Price Please inquire
Product RFP-Trap Dynabeads Kit Size 500 µL (20 reactions) Code rtdk-20 Price $ 687.5
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Product RFP VHH, recombinant binding protein Size 250 µL Code rt-250 Price $ 275
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Affinity
Dissociation constant KD of 5 nM

Wash buffer compatibility
0.5 M NaCl, 10 mM DTT, 2 % Nonidet P40 Substitute, 2 % Triton X-100

Specificity (selection)
mRFP, mCherry, mRFPruby, mPlum, tagRFP, mKate2, mOrange, PA-mCherry, mScarlet and more.
For complete list, please click here: Fluorescent protein specificity table

Binding capacity
10 µL slurry bind ~ 0.5 µg of recombinant mCherry

Coupled Nanobody/ VHH
Recombinant, monoclonal anti-Red Fluorescent Protein (RFP) single domain antibody (sdAb) fragment

Bead properties
Bead size: 2.8 µm
Storage buffer: 1x PBS, Preservative: 0.09 % sodium azide

Elution
- SDS sample buffer
- 0.2 M glycine pH 2.5
Instead of elution, we recommend on-bead assays like on-bead digestion for MS analysis.

Compatibility with mass spectrometry
The RFP-Trap is optimized for on-bead digestion. Complete tryptic digest results in 6-7 peptides.

How to cite this product
RRID: AB_2861253

Storage instructions
Shipped at ambient temperature. Upon receipt store at 4°C. Stable for one year.

What are the biophysical parameters of the RFP-Trap®?

Molecular weight: 14,9 kDa; Extinction coefficient: 30035 M-1 cm-1

Should I use an N-terminal or C-terminal fusion tag?

Both the N-terminal or C-terminal fusion tag work well with the RFP-Trap.

How can I avoid unspecific protein interactions binding to the trap?

Please find more information in our Troubleshooting guide.

How can I elute bound proteins from a trap in their native state?

You can elute your fusion protein of interest with 0.2 M glycine pH 2.5 at room temperature. Pipette the beads up and down for 60-120 seconds and repeat this step. Ensure to neutralize your supernatant immediately afterwards by adding 1 M Tris base pH 10.4.

How many mammalian cells are required for an immunoprecipitation reaction?

For one immunoprecipitation reaction, we recommend using ~10^6 - 10^7 mammalian cells. The yield is also dependent on the expression level of your protein of interest and the interaction partners.

How much cell extract should I use for an immunoprecipitation reaction?

For other type of cells than mammalian cells, we recommend using 0.5 - 1.0 mg of cell extract.

Do I need to elute bound proteins from the beads for mass spectrometry analysis?

No, you can directly conduct an on-bead digestion after immunoprecipitation. This procedure allows faster and more efficient sample preparation and a potential higher yield.
Please find more information here:

Preomics kit

Protocol on-bead digestion

Do I need to elute my protein of interest from the beads for enzymatic assays?

No, you can directly perform your enzymatic assay on the beads if the active center is not blocked.

Please find more information in our application note "enzymatic activity assay"

What are the dissociation constants of the Nano-Traps?

Generally heavy chain antibodies do have high affinities to their antigens with dissociation constants in the low nanomolar down to the picomolar range. ChromoTek has determined the following KD values:
GFP-Trap:  1 pM, picomolar (10-12 molar)*
RFP-Trap:  5 nM, nanomolar (10-9 molar)
MBP-Trap:  4 nM, nanomolar (10-9 molar)
GST-Trap:  1 nM, nanomolar (10-9 molar)*
Myc-Trap (with 2x Myc peptide): 0.5 nM, nanomolar (10-9)
Spot-Trap:  6 nM, nanomolar (10-9 molar)
*Kinetic parameter has been measured using the switchSENSE technology using electro-switchable nanolevers to analyze molecular interactions. switchSENSE is a proprietary technology from Dynamic Biosensors (www.dynamic-biosensors.com).

What is the amount of trap slurry I need for one immunoprecipitation reaction?

25 µL slurry are sufficient for one pull-down reaction as the affinity of the traps is very high.

Specifications of RFP-Trap Agarose, Magnetic Agarose, and Dynabeads

 

RFP-Trap

 

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

RFP VHH

RFP VHH

RFP VHH

RFP-tagged protein size*

Small to large size

Small to large size

Small to very large size; no size limitation

Color

White

Black

Brown

Medium particle size

90 µm

40 µm

2.8 µm

Binding capacity

9 µg/ 10 µL

8 µg/ 10 µL

0.5 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

Should I pre-clear my sample when using RFP-Trap Dynabeads?

The Dyanbeads matrix is inert and shouldn’t bind background proteins. Hence, unconjugated Dynabeads shouldn’t be used for preclearing. To investigate unspecific binding to RFP-Trap Dynabeads, we recommend to perform the IP with mock cell lysate without RFP-fusion or with RFP/mCherry only.

Benefits

  • IP and Co-IP of RFP-tagged proteins without size limitation
  • Automation and high throughput applications
  • Magnetic separation with easy and efficient washing of RFP-Trap Dynabeads
  • Very high affinity (KD=5 nM) to bind even low abundant proteins
  • No heavy & light antibody chains, short incubation (5-30 min)
  • Extraordinary binding, also under harsh conditions

Why protein size matters in immunoprecipitation

The immunoprecipitation of very large proteins like oligomers or complexes and the Co-IP of bulky/multiple binding partners may be challenging when using porous beads. The pores of agarose or magnetic agarose beads have a certain size and hence may exclude proteins, multimers, or complexes to diffuse into the pores and to interact with the Nanobody ligand; next to size also the protein’s shape may limit diffusion. Therefore, binding can just occur on the outer surface of the agarose or magnetic agarose beads, which results in a poor immunoprecipitation performance. 
Dynabeads are non-porous, solid particles with ligands coupled on their surface. Hence all size RFP-tagged proteins including very large RFP-tagged proteins, oligomers and complexes including bulky binding partners bind to the RFP Nanobody of RFP-Trap Dynabeads and are effectively immunoprecipitated.

RFP-Trap Dynabeads Kit

The RFP-Trap Dynabeads is also available in a kit, including:

  • RFP-Trap Dynabeads
  • lysis*, wash and elution buffers

*The lysis buffer provided is suitable for mammalian cells. For other cell types use another suitable lysis buffer.

Which RFP-Trap should I use?

  • RFP-Trap Agarose, when lowest background and high binding capacity IP is needed. 
  • RFP-Trap Magnetic Agarose, when magnetic separation and high binding capacity IP is needed.
  • RFP-Trap Dynabeads, when very large proteins/complexes are investigated, and magnetic separation is needed for IP.
  RFP-Trap Agarose  RFP-Trap Magnetic Agarose  RFP-Trap Dynabeads 
Low background  +++  ++  ++
Binding capacity  +++  +++  +
Size RFP-tagged protein*   Small to large  Small to large  Small to very large
Bead separation  Centrifugation  Magnetic  Magnetic

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

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Only for research applications, not for diagnostic or therapeutic use!