GFP-Catcher

Catalog No. ABIN5311508

Product Details

Key Features:

• High-affinity anti-GFP single-domain antibody (sdAb) covalently bound to 4 % cross-linked agarose beads with 50-150 µm diameter.
• The Alpaca sdAb clone 1H1 is specific for GFP and many GFP derivatives. Capacity > 3 μg GFP per μl of packed beads.
• Compatible with physiological buffers, common Lysis and Wash Buffers, and high stringency buffers.

Target: GFP (Green Fluorescent Protein (GFP))

Reactivity: Aequorea victoria

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

Purpose: GFP-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 GFP (green fluorescent protein) and common GFP derivatives like EGFP, mEGFP, Sirius, tSapphire, Cerulean, eCFP, mTurquoise, acGFP, Emerald, superecliptic pH luorin, paGFP, superfolder GFP, eYFP, mVenus and Citrine and most common CFP and YFP variants.

Cross-Reactivity (Details): Does not cross-react with mCherry, mRFP, dsRed, mTagBFP or their most common derivatives.

Characteristics: GFP-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.
GFP-Catcher thus features high affinity and superior capacity for GFP fusion proteins while showing negligible non-specific background.
GFP-Catcher immobilizes a wide range of GFP derivatives.
GFP-Catcher is compatible not only with physiological buffers but also with high stringency buffers.
GFP-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

Product Specific Information:

Our GFP Catcher is a product based on the GFP pull-down technique used to isolate GFP fusion proteins from cell lysates. It consists of special magnetic beads (product ABIN7272855) or agarose beads (product ABIN5311508) coated with an antibody against GFP. These beads allow efficient and selective binding to GFP or GFP-fused target proteins.

The application of GFP Catcher is similar to the GFP pull-down method. First, the protein of interest is expressed in the cells with it fused to GFP. Then, the cell lysate is prepared to extract the target proteins. The GFP catcher beads are then added to the cell lysate and bound to these proteins by specifically binding to the GFP or GFP fusion protein.

After the beads with the bound target proteins are isolated, thorough purification is performed to remove components that are not specifically bound. Finally, the target proteins can be separated from the beads and used for further analysis such as immunoblotting or mass spectrometry.

GFP Catcher offers the advantage of quick and easy isolation of GFP fusion proteins. It is a useful tool for protein analysis and allows efficient identification of proteins interacting with GFP fusion protein. GFP Catcher is widely used in cell biology research to study protein-protein interactions and deepen the understanding of cellular processes.

Application Details

Protocol:

This protocol provides a general outline of how to use GFP-Catcher (agarose beads) for immunoprecipitation using a microcentrifuge for sedimentation. Alternatively, it is possible to use GFP-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: GFP-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 GFP-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 GFP-Catcher (agarose beads).
8. Centrifuge GFP-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 GFP-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 GFP-Catcher (agarose beads) in 1 mL Lysis Buffer.
14. Centrifuge GFP-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 GFP-Catcher (agarose beads) gently in 1 mL TBS.
17. Centrifuge GFP-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
18. Resuspend GFP-Catcher (agarose beads) gently in 1 mL TBS.
19. Centrifuge GFP-Catcher (agarose beads) for 1 min at 3000 x g and carefully remove the supernatant.
20. Resuspend GFP-Catcher (agarose beads) resin in 50 µL 2X SDS samples buffer.
21. Heat GFP-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 GFP-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

Target Details

Target: GFP (Green Fluorescent Protein (GFP))

Alternative Name: GFP (GFP Products)

Synonyms: green fluorescent protein Bead, gfp Bead