Spot-Trap Magnetic Agarose

Description

Spot-Trap® Magnetic Agarose is an affinity reagent for immunoprecipitation (IP) of Spot-tagged proteins.
It consists of an anti-Spot-Tag® VHH/ Nanobody that is coupled to magnetic agarose beads.

Specificity
Spot-Tag sequence PDRVRAVSHWSS at the N-terminus or the C-terminus of the fusion protein.

Applications
Immunoprecipitation/ Co-IP
Mass spectrometry
ChIP/ RIP analysis
On-bead enzyme assays

Product Size Code Price Buy
Product Spot-Trap Magnetic Agarose Size 250 µL (10 reactions) Code etma-10 Price $ 275
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Product Spot-Trap Magnetic Agarose Size 500 µL (20 reactions) Code etma-20 Price $ 475
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Product Spot-Trap Magnetic Agarose Size 2.5 mL (100 reactions) Code etma-100 Price $ 2050
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Product Spot-Trap Magnetic Agarose Size 5 mL (200 reactions) Code etma-200 Price Please inquire
Product Spot-Trap Magnetic Agarose Size 10 mL (400 reactions) Code etma-400 Price Please inquire
Product Spot-Trap Magnetic Agarose Kit Size 500 µL (20 reactions) Code etmak-20 Price $ 595
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Product Spot Peptide Size 1 mg Code ep-1 Price $ 85
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Product Binding Control Magnetic Agarose Beads Size 500 µL (20 reactions) Code bmab-20 Price $ 75
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Product Spot VHH, recombinant binding protein Size 250 µL Code etx-250 Price $ 275
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Antibody
Recombinant, monoclonal single domain antibody (sdAb) fragment

Affinity
Dissociation constant KD of 6 nM

Wash buffer compatibility
2 M urea, 2 M NaCl, 10 mM DTT, 2 % Nonidet P40 Substitute, 1 % Triton X-100, 0.1 % SDS

Specificity
Spot-Tag sequence motif PDRVRAVSHWSS

Binding capacity
10 µL slurry bind ~ 7 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: ~ 40 µm
Storage buffer: 20 % EtOH

Elution
- Spot peptide
- 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.

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

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 elute bound Spot-tagged protein from the Spot-Trap in its native state?

You can elute your Spot-fusion competitively with Spot peptide. Alternatively, you can use 10 mM NaOH pH 12 (adjust pH immediately after elution).

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: etx-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 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 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.

Benefits

  • NO contaminating heavy and light antibody chains
  • Short incubation time of 30-60 minutes
  • Just 4-5 peptides in mass spec.
  • Recombinantly expressed without batch-to-batch variation
  • Well-characterized and validated
  • Ready to use

Spot-Trap Magnetic Agarose Kit

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

  • Spot-Trap Magnetic Agarose
  • Lysis*, wash and elution buffers

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

Binding Control for Magnetic Agarose Beads

Spot Peptide

Elution of native and functional Spot-tagged fusion proteins with Spot peptide.
Spot peptide has the amino acid sequence PDRVRAVSHWSS.

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