BFP-Catcher

Catalog No. ABIN5311512

Product Details

Target: Blue Fluorescent Protein (BFP)

Reactivity: Entacmaea quadricolor

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

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

Sample Type: Cell Extracts

Specificity: Recognizes mTagBFP, mKate, mKate2, mTagRFP, mTagRFP657 and most common fluorescent proteins deriving from Entacmaea quadricolor

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

Characteristics: BFP-Catcher is based on a high-affinity single-domain antibody (sdAb) that is covalently immobilized on 4 % cross-linked 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. BFP-Catcher thus features high affinity and superior capacity for BFP fusion proteins while showing negligible non-specific background.
BFP-Catcher is compatible not only with physiological buffers but also with high stringency buffers.
BFP-Catcher thus provides great freedom to adjust the binding and washing conditions to the experimental needs.

Components: 4 % cross-linked agarose (bead size 50-150 μm) with covalently immobilized single-domain antibody

Material not included: wash buffers, columns, tubes

Bead Ligand: Antibody

Bead Matrix: Agarose beads

Bead Size: 90 μm

Application Details

Application Notes: Coating: sdAb anti-BFP clone 1H7
Matrix: 4 % cross-linked agarose, bead size 50-150 μm
Capacity: > 3 μg BFP per μl of packed beads
Buffer Compatibility:

• Common buffer substances at pH 5 to 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
• RNAse A, DNAse I, Benzonase, protease inhibitors

Protocol:

This protocol provides a general outline of how to use BFP-Catcher (agarose beads) for immunoprecipitation using a microcentrifuge for sedimentation. Alternatively, it is possible to use BFP-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: BFP-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 BFP-Catcher (agarose 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 BFP-Catcher (agarose beads).
8. Centrifuge BFP-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
9. Repeat wash steps once for a total of two washes.
10. Resuspend equilibrated BFP-Catcher (agarose beads) gently with the cell lysate supernatant.
11. Rotate the microcentrifuge tubes for 1 h at 4 °C.
12. Centrifuge microcentrifuge tubes for 1 min at 1000 x g at 4 °C. Keep a small sample as “unbound” fraction. Carefully remove the supernatant.
13. Resuspend BFP-Catcher (agarose beads) in 1 mL Lysis Buffer.
14. Centrifuge BFP-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
15. Repeat wash steps twice for a total of three washes.
16. Resuspend BFP-Catcher (agarose beads) gently in 1 mL TBS.
17. Centrifuge BFP-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
18. Resuspend BFP-Catcher (agarose beads) gently in 1 mL TBS.
19. Centrifuge BFP-Catcher (agarose beads) for 1 min at 3000 x g and carefully remove the supernatant.
20. Resuspend BFP-Catcher (agarose beads) resin in 50 µL 2X SDS samples buffer.
21. Heat BFP-Catcher (agarose beads) resin for 5 min to 95 °C.
22. Centrifuge microcentrifuge tubes for 1 min at 3000 x g and transfer the supernatant to fresh microcentrifuge tubes. Keep the BFP-Catcher (agarose 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

References

Devant, Boršić, Ngwa, Xiao, Chouchani, Thiagarajah, Hafner-Bratkovič, Evavold, Kagan: "Gasdermin D pore-forming activity is redox-sensitive." in: Cell reports, Vol. 42, Issue 1, pp. 112008, (2023) (PubMed).

Target Details

Target: Blue Fluorescent Protein (BFP)

Alternative Name: TagBFP (BFP Products)