Spot-Trap Dynabeads

Description
Spot-Trap® Dynabeads are affinity beads for immunoprecipitation (IP) of Spot-tagged proteins
It comprises an anti-Spot-Tag® VHH/ Nanobody coupled to DynabeadsTM.

More information about the Spot capture and detection tag can be found here.

Specificity
Spot-Tag sequence PDRVRAVSHWSS

Applications
Immunoprecipitation/ Co-IP
Mass spectrometry
ChIP/ RIP analysis
On-bead enzyme assays
For protein purification use Spot-Cap

When use Spot-Trap? When use Spot-Cap? Click here.

Product Size Code Price Buy
Product Spot-Trap Dynabeads Size 250 µL (10 reactions) Code etd-10 Price $ 335
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Product Spot-Trap Dynabeads Size 500 µL (20 reactions) Code etd-20 Price $ 550
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Product Spot-Trap Dynabeads Size 2.5 mL (100 reactions) Code etd-100 Price $ 2475
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Product Spot-Trap Dynabeads Size 5 mL (200 reactions) Code etd-200 Price Please inquire
Product Spot-Trap Dynabeads Size 10 mL (400 reactions) Code etd-400 Price Please inquire
Product Spot-Trap Dynabeads Kit Size 500 µL (20 reactions) Code etdk-20 Price $ 715
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Product Spot Peptide Size 1 mg Code ep-1 Price $ 85
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Product Spot VHH, recombinant binding protein Size 250 µL Code etb-250 Price $ 455
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Affinity
Dissociation constant KD of 6 nM

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

Specificity
Spot-Tag sequence motif PDRVRAVSHWSS

Binding capacity
10 µL slurry bind about 0.9 µg of recombinant Spot-Tag fusion protein (30 kDa)

Coupled Nanobody/ VHH
Recombinant, monoclonal anti-Spot-Tag 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
- 10 mM NaOH pH 12
Please consider whether elution is really needed. We recommend on-bead assays like on-bead digestion for MS analysis.
For protein purification and efficient elution with Spot peptide at +4°C we recommend Spot-Cap™ affinity resin.

Compatibility with mass spectrometry
The Spot-Trap is optimized for on-bead digestion. Complete tryptic digest results in 4-5 peptides.

How to cite this product
RRID: AB_2861252

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

Should I use an N-terminal or C-terminal Spot-fusions? Can I also insert the Spot-Tag in the middle of my protein?

Both, an N- or C-terminal fusion work well.
The use of the Spot-Tag for internal protein tagging has to be tested case by case. The Spot-Tag peptide has to exist in a linear form and be accessible without steric hindrance from other parts of the protein of interest. An internal Spot-Tag is only likely to be recognized by the Spot-Tag nanobody if inserted into a sufficiently large and unstructured loop, an inherently unstructured domain or a lengthy domain linker.

How can I detect my Spot-fusion in Western blot application?

You can use ChromoTek's Spot-Label to detect your Spot-tagged fusion protein. Alternatively, you can use ChromoTek's Spot-Binding protein (Spot VHH, product code: etb-250) followed by incubation with a conventional anti-Llama or anti-His6 secondary antibody.

How long should I incubate my Spot-fusion sample with Spot-Label for Western blot and IF applications?

Optimal results are achieved through overnight incubation.

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

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

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

For preclearing of your sample we recommend to use our binding controls (bab-20 or bmab-20).

Please find more information in our Troubleshooting guide

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 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.

How can I elute bound Spot-tagged protein from the Spot-Trap?

You can elute your Spot-fusion with 2xSDS-sample buffer or you can use 10 mM NaOH pH 12 (adjust pH immediately after elution). For protein purification and efficient elution with Spot peptide at +4°C we recommend Spot-Cap™ affinity resin.

Should I preclear my sample when using Spot-Trap Dynabeads?

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

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

 

Spot-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

Spot VHH

Spot VHH

Spot VHH

Spot-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**

7 µg/ 10 µL

7 µg/ 10 µL

0.9 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

** Determiend with a recombinant Spot-Tag fusion protein (30 kDa)

 

Benefits

  • Magnetic separation with easy and efficient washing of Spot-Trap Dynabeads
  • IP and Co-IP of Spot-tagged proteins without size limitation
  • NO contaminating heavy and light antibody chains
  • Just 4-5 peptides in mass spec.
  • Recombinantly expressed without batch-to-batch variation
  • Well-characterized and validated
  • Ready to use
  • Short incubation time of 30-60 minutes

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 Spot-tagged proteins including very large Spot-tagged proteins, oligomers and complexes including bulky binding partners bind to the Spot Nanobody of Spot-Trap Dyanbeads and are effectively immunoprecipitated.

Spot-Trap Dynabeads Kit

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

  • Spot-Trap Dynabeads
  • Lysis* and wash buffers

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

Which Spot-Trap should I use?

  • Spot-Trap Agarose, when lowest background and high binding capacity IP is needed. 
  • Spot-Trap Magnetic Agarose, when magnetic separation and high binding capacity IP is needed.
  • Spot-Trap Dynabeads, when very large proteins/complexes are investigated, and magnetic separation is needed for IP.
  Spot-Trap Agarose  Spot-Trap Magnetic Agarose  Spot-Trap Dynabeads 
Low background  +++  ++  ++
Binding capacity  +++  +++  +
Size Spot-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

Testimonials

“Therefore, we considered that small Spot-Tag is better than large GFP-tag in terms of affecting functions of fused protein(s). The obtained IP/MS data showed many interesting candidates that may function with “protein X” in cells. We are now validating these interactions. In conclusion, Spot-Tag and Spot-Trap system works very well in IP/MS experiments.”
Associate Professor, University of Tokushima, Japan


“Mass spec results look great.  High specificity, low background, and I am seeing more interacting partners of my protein of interest than with the HA epitope tag that I have historically used.  I am very pleased with the results.”
Graduate Student, Cornell University, NY, USA


“I just finished testing the Spot-Tag. I obtained interesting results by performing a small scale purification of my fusion protein, the protein is very pure and there are no non-specific bands on the beads.”
Assistant Engineer, CNRS, Lyon, France


“The Spot-Trap test worked really well (equivalent efficiency to Twin-Strep tag)….in detergent.”
Postdoctoral researcher, University of Oxford, UK


“The Spot-Tag works well for pulldowns; much better than the Strep-tag, at least for my constructs.”
Contractor, NIH, Bethesda, MD, USA


“GFP-Trap and Spot-Trap both specifically bind Spot-GFP, and Spot-Trap appears at least as efficient as GFP-Trap.”
Senior Research Associate, University of Bristol, UK

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