RFP-Catcher (agarose magnetic beads)

Catalog No. ABIN7529450

Product Details

Target: RFP (Red Fluorescent Protein (RFP))

Reactivity: Discosoma

Application: RNA-Binding Protein Immunoprecipitation (RIP), Protein Complex Immunoprecipitation (Co-IP), Immunoprecipitation (IP), Purification (Purif), Chromatin Immunoprecipitation (ChIP)

Purpose: RFP-Catcher is based on a high-affinity single-domain antibody (sdAb) that is covalently immobilized on 4% cross-linked magnetic agarose.

Specificity: MRFP (red fuorescent protein) and other derivatives like mOrange, dsRed1, dsRED2, tdTomato, mCherry and mScarlet-i.

Cross-Reactivity (Details): Does not cross-react with GFP or mTagBFP derivatives.

Characteristics: RFP-Catcher is based on a high-affinity single-domain antibody (sdAb) that is covalently immobilized on 4 % cross-linked magnetic agarose beads. The innovative, oriented and selective attachment via a flexible linker guarantees a high accessibility of the sdAbs and largely eliminates batch-to-batch variations. Due to the single-chain nature of sdAbs and their covalent attachment, no "leakage" of light and heavy chains from IgGs is observed during elution with SDS sample buffer.
RFP-Catcher thus features high affinity and superior capacity for RFP fusion proteins while showing negligible non-specific background.
RFP-Catcher immobilizes a wide range of RFP derivatives.
RFP-Catcher is compatible not only with physiological buffers but also with high stringency buffers.
RFP-Catcher thus provides great freedom to adjust the binding and washing conditions to the experimental needs.

Bead Ligand: Antibody

Bead Matrix: Magnetic Agarose beads

Bead Size: 90 μm

Application Details

Application Notes: Capacity: > 3 μg RFP per μl of packed beads

Protocol:

This protocol provides a general outline of how to use RFP-Catcher (agarose magnetic beads) for immunoprecipitation using a microcentrifuge for sedimentation. Alternatively, it is possible to use RFP-Catcher agarose beads in spin columns. All protocol steps should be carried out at 4 °C.

1. For mammalian cells, harvest 10⁶-10⁸ cells per sample.
2. Lyse cells according to established protocols in 0.2 to 1.5 mL volume. Recommended Buffer Conditions: RFP-Catcher resins are compatible with commonly used Lysis and Washing buffers, e.g. RIPA buffer. The following buffer conditions have been tested:
   • pH ranging from pH 5 to pH 9
   • 2 % Triton X-100, 1 % Tween-20, 1 % NP-40, 1 % CHAPS, 1 % Deoxycholate, 0.1 % SDS
   • 4 M NaCl, 2 M KCl, 1 M MgCl₂
   • 100 mM EDTA
   • 4 M urea
   • 10 mM DTT, 10 mM 2-Mercaptoethanol
   • Protease Inhibitors
   • RNAse A, DNAse I, Benzonase
3. Centrifuge cell lysates in microcentrifuge tubes for 10 min at 14.000 x g at 4 °C. Keep a small samples as “input” fraction.
4. Transfer the supernatant to a fresh microcentrifuge tube for each sample and keep at 4 °C.
5. Homogenize the RFP-Catcher (agarose magnetic beads) slurry gently by shaking.
6. Transfer 20 μL bead slurry to a 1.5 mL microcentrifuge tube for each sample.
7. Add 1 mL Lysis Buffer to equilibrate RFP-Catcher (agarose magnetic beads).
8. Place the tubes on a magnet stand until the fluid is clear. Remove the supernatant carefully.
9. Repeat wash steps once for a total of two washes.
10. Resuspend equilibrated RFP-Catcher (agarose magnetic beads) gently with the cell lysate supernatant.
11. Rotate the microcentrifuge tubes for 1 h at 4 °C.
12. Place the tubes on a magnet stand until the fluid is clear. Keep a small aliquot of the supernatant as “unbound” fraction. Carefully remove the supernatant.
13. Resuspend RFP-Catcher (agarose magnetic beads) in 1 mL Lysis Buffer.
14. Place the tubes on a magnet stand until the fluid is clear and carefully remove the supernatant.
15. Repeat wash steps twice for a total of three washes.
16. Resuspend RFP-Catcher (agarose magnetic beads) gently in 1 mL TBS.
17. Place the tubes on a magnet stand until the fluid is clear and carefully remove the supernatant.
18. Repeat wash steps once for a total of two washes.
19. Resuspend RFP-Catcher (agarose magnetic beads) resin in 50 µL 2X SDS samples buffer.
20. Heat RFP-Catcher (agarose magnetic beads) resin for 5 min to 95 °C.
21. Place the tubes on a magnet stand until the fluid is clear and transfer the supernatant to fresh microcentrifuge tubes. Keep the RFP-Catcher (agarose magnetic beads) as backup.

Restrictions: For Research Use only

Handling

Buffer: 50 % slurry in PBS containing 20 % Ethanol

Storage: 4 °C

Storage Comment: Store at 4 °C, Do not freeze!

Expiry Date: 12 months

Target Details

Target: RFP (Red Fluorescent Protein (RFP))

Alternative Name: RFP (RFP Products)