Spot-Trap® Agarose is an affinity bead for immunoprecipitation (IP) and affinity purification of Spot-tagged proteins
It comprises an anti-Spot-Tag® VHH/ Nanobody coupled to agarose beads.
Spot-Tag sequence PDRVRAVSHWSS at the N-terminus or the C-terminus of the fusion protein.
ChIP/ RIP analysis
On-bead enzyme assays
|Product Spot-Trap Agarose||Size 250 µL (10 reactions)||Code eta-10||Price $ 275||Buy +|
|Product Spot-Trap Agarose||Size 500 µL (20 reactions)||Code eta-20||Price $ 475||Buy +|
|Product Spot-Trap Agarose||Size 2.5 mL (100 reactions)||Code eta-100||Price $ 2050||Buy +|
|Product Spot-Trap Agarose||Size 5 mL (200 reactions)||Code eta-200||Price Please inquire|
|Product Spot-Trap Agarose||Size 10 mL (400 reactions)||Code eta-400||Price Please inquire|
|Product Spot-Trap Agarose Kit||Size 500 µL (20 reactions)||Code etak-20||Price $ 595||Buy +|
|Product Spot Peptide||Size 1 mg||Code ep-1||Price $ 85||Buy +|
|Product Binding Control Agarose Beads||Size 500 µL (20 reactions)||Code bab-20||Price $ 75||Buy +|
|Product Spin Columns||Size 10 units||Code sct-10||Price $ 35||Buy +|
|Product Spin Columns||Size 20 units||Code sct-20||Price $ 60||Buy +|
|Product Spin Columns||Size 50 units||Code sct-50||Price $ 130||Buy +|
|Product Spot VHH, recombinant binding protein||Size 250 µL||Code etb-250||Price $ 455||Buy +|
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
Spot-Tag sequence motif PDRVRAVSHWSS
10 µL slurry bind about 7 µg of recombinant Spot-Tag fusion protein (30 kDa)
Coupled Nanobody/ VHH
Recombinant, monoclonal anti-Spot-Tag single domain antibody (sdAb) fragment
Bead size: ~ 90 µm (cross-linked 4 % agarose beads)
Storage buffer: 20 % EtOH
- 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.
Shipped at ambient temperature. Upon receipt store at 4°C. Stable for one year.
Protocol for immunoprecipitation
Protocol for affinity purification
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?
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?
Do I need to elute my protein of interest from the beads for enzymatic assays?
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.
- 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
Spot-Trap Agarose Kit
The Spot-Trap Agarose is also available in a kit, including:
- Spot-Trap 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 Agarose Beads
“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
Only for research applications, not for diagnostic or therapeutic use!