iST RFP-Trap Kit

Description

Kit for immunoprecipitation (IP) and Co-immunoprecipitation (Co-IP) of RFP- and mCherry fusion proteins & sample preparation for mass spectrometry (MS)

Specificity
mCherry, mRFP, mRFPruby, mPlum, tagRFP, mKate2 and many more RFP derivatives, see: Fluorescent protein specificity table

Applications
Identification of RFP-fusion proteins & complexes by MS
Interactome studies
Intuitive protocol and reproducible results
Superior processing of IP samples into clean peptide mixtures

Details
The iST RFP-Trap Kit for processing RFP-fusion proteins and their interacting partners for mass spectrometry analysis: The kit includes the RFP-Trap for IP/Co-IP of RFP-fusion 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 RFP-Trap Kit Size 8 reactions (plus 2 controls) Code rtak-iST-8 Price $ 595
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Affinity
Dissociation constant KD of 5 nM

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 more than 9 µg of recombinant mCherry

Coupled Nanobody/ VHH
Recombinant, monoclonal anti-Red Fluorescent Protein (RFP) 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 RFP-Trap is optimized for on-bead digestion. Complete tryptic digest results in 6-7 peptides

Kit components

Component

Description

RFP-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.

 

 

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 RFP-Trap at 4°C and Digest at -20°C.

What is the dissociation constant of the RFP-Trap?

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:
RFP-Trap:          5 nM, nanomolar (10-9 molar)

How can I avoid unspecific protein interactions binding to the RFP-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

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.

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.

Details

The iST RFP-Trap Kit enables researchers to process RFP-fusion proteins and their interacting partners for Mass Spectrometry analysis by including the ChromoTek RFP-Trap for immunoprecipitation/Co-IP of RFP-fusion 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 RFP fusion proteins & sample preparation for MS

  • Reliable dentification of RFP-fusion proteins & interacting partners by MS
  • Identification of even low abundant RFP-fusion proteins
  • Superior processing of IP samples into clean peptide mixtures
  • Intuitive protocol for reproducible results

RFP-Trap Agarose

The ChromoTek RFP-Trap is an affinity resin for immunoprecipitation of RFP-fusion proteins. It consists of an RFP Nanobody/ VHH coupled to agarose beads.

  • Fast and efficient one-step IP
  • More than 120 publications

iST Technology

The PreOmics iST technology provides a complete solution for proteomic sample preparation.

  • Streamlined workflow directly compatible with MS analysis
  • Avoids MS downtime through clean peptide samples
  • Developed at the laboratory of Matthias Mann, MPI for Biochemistry

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 RFP-Trap Kit content

The iST RFP-Trap Kit contains sufficient material for up to 10 immunoprecipitations. Out of these ten reactions, two shall be used for negative/positive controls by immunoblot analysis. The iST RFP-Trapt Kit can be used for up to 8 iST proteomic sample preparation reactions.

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