Immunoprecipitation Protocol Using Anti-GFP Antibodies

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile marker for monitoring physiological processes, visualizing protein localization, and detecting transgenic expression.^1-5 We offer the anti-GFP antibody as a rabbit polyclonal, monoclonal, or IgG fraction, two mouse monoclonal antibodies, and a chicken IgY fraction.  All six anti-GFP antibodies are suited for detection of native GFP, GFP variants, and most GFP fusion proteins by western blot analysis while the rabbit and mouse antibodies are also useful for immunoprecipitation.  A summary of the validated applications for all of these antibodies can be found in Table 1.

The anti-GFP rabbit polyclonal antibody is raised against GFP isolated directly from Aequorea victoria and is available as a complete antiserum (A6455) or as an IgG fraction purified by ion-exchange chromatography (A11122).

Like our multi-purpose polyclonal anti-GFP antibody, the rabbit anti-GFP monoclonal antibody (G10362) is raised against full-length GFP; it is suitable for immunoprecipitation, immunohistochemistry, and western blotting.

Anti-GFP mouse monoclonal antibody 3E6 (A11120) is useful for immunoprecipitation, immunocytochemical localization, and immunosorbent assays (ELISA).

Anti-GFP mouse monoclonal antibody 11E5 (A11121) is optimized for western analysis, allowing colorimetric detection of as little as 10 ng of GFP or GFP-fusion proteins, or chemiluminescent detection of picogram quantities.

The chicken anti-GFP antibody (A10262) is raised against GFP isolated directly from Aequorea victoria (Table 1), and the IgY fraction is purified by affinity purification. The chicken IgY lacks a classic “Fc” domain and does not bind to mammalian IgG Fc receptors, resulting in lower backgrounds during immunostaining protocols. The chicken IgY is also antigenically different from the mammalian IgG, allowing you to perform double immunostaining experiments using antibodies from multiple species.

Table 1. Anti-GFP Antibodies and Their Applications.

*  2 mg/mL. # Immunoprecipitation (IP), immunohistochemistry (IHC), western blot (WB), immunocytochemistry (ICC), and flow cytometry (Flow).

• 1X Phosphate-buffered saline, pH 7.4 (PBS, Cat. no. 10010-031)
• Blocking buffer: 5% Normal Goat Serum (NGS) in PBS, pH 7.4
• Wash buffer: 5% NGS in PBS, 1% Triton® X-100, 3% Bovine Serum Albumin (BSA)
• Magnetic rack
• NuPAGE® LDS Sample Buffer (Cat. no. NP0007) or an equivalent SDS sample buffer
• NuPAGE® gel or equivalent SDS gel
• 
  

• Sheep anti-rabbit magnetic beads (Cat. no. 112-03)
• Sheep anti-mouse magnetic beads (Cat. no. 110-31)

1. Transfer the required amount of beads to a clean microcentrifuge tube.


2. Place the tubes containing beads on a magnetic rack for 1 minute. Carefully discard the supernatant.


3. Resuspend the beads in 500 µL of PBS by pipetting gently up and down. Remove the tubes from the magnetic rack and rotate the tubes gently for 10 minutes.


4. Place the tubes on a magnetic rack for 1 minute and discard the supernatant.


5. Repeat the wash steps 1.3–1.4 two more times. Resuspend the beads in 500 µL of PBS. Do not allow the beads to dry out.

1. Preclear the lysate by combining 50 µL of sheep anti-rabbit or sheep anti-mouse magnetic beads with 30 µg of sample lysate in a microcentrifuge tube. Incubate the tube at 4°C with gentle rotation for 1 hour.


2. Place tubes on a magnetic rack for 1 minute and transfer the supernatant into a new microcentrifuge tube placed on ice.


3. To the precleared lysate, add 5% NGS in PBS for blocking, and then add primary anti-GFP antibody diluted in PBS to a final concentration of 0.2 µg/mL. Incubate the sample at 4°C overnight with gentle rotation.


4. Place the tubes containing the sheep anti-rabbit (for rabbit antibody) or sheep anti-mouse (for mouse antibody) magnetic beads (prepared in steps 1.1–1.5) on a magnetic rack for 1 minute and discard the supernatant. Add the sheep anti-rabbit or sheep anti-mouse magnetic beads to the samples. Incubate the samples at room temperature for 1 hour with gentle rotation.


5. Place the tubes on a magnetic rack for 1 minute. Discard the supernatant.


6. Remove the tubes from the magnetic rack, and resuspend the beads in Wash buffer (5% NGS in PBS, 1% Triton® X-100, 3% BSA) by pipetting gently up and down. Rotate the tubes gently for 10 minutes.


7. Place the tubes on a magnetic rack for 1 minute. Discard the supernatant.


8. Repeat the wash steps 2.6–2.7 two more times.


9. Add 25 µL of 1X SDS sample buffer to the beads. Heat the samples at 100°C for 5 minutes. If you are using NuPAGE® LDS Sample Buffer, heat the samples at 70°C for 10 minutes.


10. Place the tubes on a magnetic rack for 1 minute. Transfer the supernatant to a clean, microcentrifuge tube, and analyze the supernatant using SDS-PAGE.