iST Spot-Trap Kit
3
Description
Kit for IP/ Co-IP of Spot-tagged proteins & sample preparation for mass spectrometry (MS)
Specificity
Spot-tag sequence motif PDRVRAVSHWSS.
Applications
Identification of Spot-tagged proteins & interacting partners by MS
Interactome studies
Superior processing of IP samples into clean peptide mixtures
Intuitive protocol for reproducible results
Details
The iST Spot-Trap Kit enables researchers to process Spot-tagged proteins and their interacting partners for Mass Spectrometry analysis by including the ChromoTek Spot-Trap for immunoprecipitation/Co-IP of Spot-tagged proteins and the PreOmics iST buffers and cartridges required for bottom-up proteomic sample preparation. This robust method yields purified peptides while dramatically reducing contamination and sample loss. Each kit accommodates up to eight samples and includes pull-down reagent for controls.
| Product | Size | Code | Price | Buy |
|---|---|---|---|---|
| Product iST Spot-Trap Kit | Size 8 reactions (plus 2 controls) | Code etak-iST-8 | Price $ 595 |
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Affinity
Dissociation constant KD of 6 nM
Specificity
Spot-tag sequence motif PDRVRAVSHWSS
Binding capacity
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 properties
Bead size: ~ 90 µm (cross-linked 4 % agarose beads)
Storage buffer: 20 % EtOH
Compatibility with mass spectrometry
The Myc-Trap is optimized for on-bead digestion. Complete tryptic digest results in 4-5 peptides
Kit components
Component | Description |
Spot-Trap Agarose | 8 reactions plus 2 controls |
Digest | Enzyme Trypsin-mix to digest proteins. |
Resuspend buffer | Protease reconstitution buffer for enzymes. |
Lyse buffer | Denature, reduce and alkylate proteins. |
Stop solution | Stop the enzymatic activity. |
Wash 1 buffer | Clean up peptides from hydrophobic contaminants. |
Wash 2 buffer | Clean up peptides from hydrophilic contaminants. |
Elution buffer | Elute the peptides from the cartridge. |
LC-Load load | Load peptides on reversed-phase LC-MS column. |
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Cartridges | Cartridge for 1 to 100 µg protein starting material. |
Waste tubes | Tube for collecting waste after washing steps. |
Collection tubes | Tube for collecting peptides after elution. |
Adapters | Enables placing a cartridge into a tube. |
Caps | Cap to optionally close the cartridge’s bottom. |
Storage instructions
Kit is shipped at ambient temperature. Upon receipt store Spot-Trap at 4°C and Digest at -20°C.
Protocol
SDS
Brochure
Application notes
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:
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.
Should I use an N-terminal or C-terminal fusion tag?
Both the N-terminal or C-terminal fusion tag work well with the Spot-Trap.
Details
The iST Spot-Trap Kit enables researchers to process Spot-tagged proteins and their interacting partners for Mass Spectrometry analysis by including the ChromoTek Spot-Trap for immunoprecipitation/Co-IP of Spot-tagged proteins and the PreOmics iST buffers and cartridges required for bottom-up proteomic sample preparation. This robust method yields purified peptides while dramatically reducing contamination and sample loss. Each kit accommodates up to eight samples and includes pull-down reagent for controls.
IP / Co-IP of Spot-tagged proteins & sample preparation for MS
- Reliable dentification of Spot-tagged proteins & interacting partners by mass spectrometry.
- Identification of even low abundant/ low expressed Spot-tagged proteins
- Superior processing of IP samples into clean peptide mixtures
- Intuitive protocol and reproducible results
Spot-Trap Agarose
The ChromoTek Spot-Trap is a Nanobody or VHH coupled to a matrix for immunoprecipitation of Spot-tagged proteins & their interacting partners.
- Fast and efficient one-step IP
- Low background
iST Technology
The PreOmics iST technology provides a complete solution for proteomic sample preparation.
- Developed at the laboratory of Matthias Mann, MPI for Biochemistry
- Streamlined workflow directly compatible with MS analysis
- Avoids MS downtime through clean peptide samples
Technology
MS-based proteomics typically employs multiple sample preparation steps that can lead to sample contamination and loss. The PreOmics iST technology overcomes these problems and has been published in Nature Methods (Kulak et al., 2014): “Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells” (doi:10.1038/nmeth.2834).
The PreOmics iST technology includes all chemicals, enzymes, cartridges and plasticware required for denaturation, reduction, and alkylation of co-immunoprecipitated proteins plus in-solution digestion and peptide clean-up. Optimized and patented peptide washing procedures eliminate both hydrophobic and hydrophilic contaminants. This results in clean peptides, decreasing MS downtime, and providing reproducible and reliable results. For more information see www.preomics.com.
iST Spot-Trap Kit content
The iST Spot-Trap Kit contains sufficient Spot-Trap Agarose for up to 10 immunoprecipitations. Out of these ten reactions, two shall be used for negative/positive controls by immunoblot analysis. The iST Spot-Trap Kit can be used for up to 8 iST proteomic sample preparation reactions.
Only for research applications, not for diagnostic or therapeutic use!