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Introduction

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 Invitrogen offers 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.

Catalog No.
Host & Type
Amount
Application# Form
A6455Rabbit Polyclonal
100 µL
IP, IHC, WB
Serum
A10262Chicken Polyclonal
100 µL*ICC, WB
IgY Fraction
A11122Rabbit Polyclonal
100 µL*IP, IHC, WB
IgG Fraction
A11120Mouse Monoclonal
100 µgIP, IHC
IgG2a
A11121
Mouse Monoclonal100 µgWB
IgG1
G10362
Rabbit Monoclonal
100 µgELISA, IP, IHC, WB, Flow
IgG

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

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Materials

Preparing the Anti-GFP Monoclonal Antibody Stock Solutions    

To prepare 0.2 mg/mL stock solutions, reconstitute the lyophilized antibodies in 0.5 mL of phosphate-buffered saline (PBS), pH 7.4. You may store these solutions for up to 3 months at 4°C with the addition of 2 mM sodium azide.

Use the following immunoprecipitation protocol with rabbit and mouse anti-GFP antibodies but not with chicken anti-GFP antibody. Read the entire protocol before starting.


Materials Required but Not Provided


  • 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

One of the following:
  • Sheep anti-rabbit magnetic beads (Cat. no. 112-03)
  • Sheep anti-mouse magnetic beads (Cat. no. 110-31)


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Ordering Information

Sku Name Size Price Qty
A6455 GFP Rabbit Serum Polyclonal Antibody 100 µL CNY 2,980.00
A11120 GFP (clone 3E6) Mouse IgG2a, Monoclonal Antibody 100 µg CNY 3,420.00
A11121 GFP (clone 11E5) Mouse IgG1, Monoclonal Antibody 100 µg CNY 3,420.00
A11122 GFP Rabbit IgG Polyclonal Antibody Fraction 100 µL CNY 4,445.00
G10362 GFP Monoclonal Antibody, ABfinity™ Recombinant 100 µg CNY 3,330.00
11031 Dynabeads® Sheep-Anti Mouse IgG 5 mL CNY 7,200.00
11203D Dynabeads® M-280 Sheep Anti-Rabbit IgG 2 mL CNY 3,870.00
14190136 DPBS, no calcium, no magnesium 1,000 mL CNY 411.00
10010031 PBS, pH 7.4 1,000 mL CNY 389.00
R10367 Red Fluorescent Protein (RFP) Rabbit Polyclonal Antibody, Purified 100 µg CNY 2,920.00

Protocol

1.  Preparation of Magnetic Beads

For each sample, you will need 50 µL of sheep anti-rabbit or sheep anti-mouse magnetic beads.

  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.


2.  Immunoprecipitation Protocol

  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.

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References

1. Methods in Enzymology, Vol. 302, P.M. Conn, Ed., Academic Press (1999).

2. Annu Rev Biochem 67, 509 (1998).

3. Nat Biotechnol 15, 961 (1997).

4. Nature 369, 400 (1994).

5. Science 263, 802 (1994).

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MP 06455   26–April–2010