GFP-Catcher

• 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 >4 µg GFP / 1 µL packed beads (2 µL bead slurry).
• Compatible with physiological buffers, common Lysis and Wash Buffers, and high stringency buffers.

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.

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.

Protocol as PDF

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.