RFP-Trap Magnetic Beads

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

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 Magnetic Beads	Size 250 µL (10 reactions)	Code rtd-10	Price $ 335	Buy +
Product RFP-Trap Magnetic Beads	Size 500 µL (20 reactions)	Code rtd-20	Price $ 550	Buy +
Product RFP-Trap Magnetic Beads	Size 2.5 mL (100 reactions)	Code rtd-100	Price $ 2475	Buy +
Product RFP-Trap Magnetic Beads	Size 5 mL (200 reactions)	Code rtd-200	Price Please inquire
Product RFP-Trap Magnetic Beads	Size 10 mL (400 reactions)	Code rtd-400	Price Please inquire
Product RFP-Trap Magnetic Beads Kit	Size 500 µL (20 reactions)	Code rtdk-20	Price $ 715	Buy +
Product RFP VHH, recombinant binding protein	Size 250 µL	Code rt-250	Price $ 275	Buy +

Affinity
Dissociation constant K_D 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/ V_HH
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.

Protocol

Brochure

RFP-Trap product brochure (PDF)

Trouble shooting

Troubleshooting guide immunoprecipitation (IP) (PDF)

SDS

RFP-Trap Specificity

Fluorescent protein specificity table (PDF)

Application notes

Chromatin Immunoprecipitation (ChIP) with RFP-tagged proteins (PDF)

White papers

Split Fluorescent Protein Technology (PDF)

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 Magnetic Beads

	RFP-Trap
	Agarose	Magnetic Agarose	Magnetic Beads
Matrix	Agarose (4% cross-linked)	Magnetic agarose (6% cross linked)	Magnetic Beads 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 Magnetic Beads?

The Magnetic Beads matrix is inert and shouldn’t bind background proteins. Hence, unconjugated Magnetic Beads shouldn’t be used for preclearing. To investigate unspecific binding to RFP-Trap Magnetic Beads, 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 Magnetic Beads
Very high affinity (K_D=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.
Magnetic Beads 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 Magnetic Beads and are effectively immunoprecipitated.