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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) detection, immunocytochemistry (ICC), and flow cytometry (Flow).
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Materials

The following protocol is designed for immunocytochemistry using the anti-GFP, chicken IgY fraction (A10262). Read the entire protocol before starting.

Materials Required but Not Provided

  • 1X Dulbecco’s Phosphate-buffered saline (D-PBS, Cat. no. 14190-136)
  • Fixative solution: 4% Formaldehyde solution in PBS, pH 7.4
  • Permeabilizing solution: 0.25% Triton® X-100 in PBS, pH 7.4
  • Blocking solution: 5% Normal Goat serum in PBS, pH 7.4
  • Conjugated secondary anti-chicken IgG antibody for detection
  • 1X Phosphate-buffered saline (PBS) pH 7.4 (Cat. no. 10010-031)

Preparing Cells

Culture mammalian cells on cover slips in the appropriate medium to ~75% confluency.
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Protocol

  1. Remove the media from the cells grown on cover slips. Rinse the cells twice for 1 minutes each in D-PBS.

  2. Fix the cells in Fixative solution (4% formaldehyde in PBS) for 30 minutes at room temperature with gentle agitation in the dark. Remove the solution.

  3. Wash the cells twice in PBS for 1 minute each with gentle agitation. Remove the PBS.

  4. Permeabilize the specimen with Permeabilization solution (0.25% Triton® X-100 in PBS) for 5 minutes at room temperature with gentle agitation in the dark. Remove the solution.

  5. Wash the cells twice in PBS for 1 minute each with gentle agitation. Remove the PBS.

  6. Add Blocking solution (5% Normal Goat Serum in PBS, pH 7.4) to the cells. Incubate for 1 hour at room temperature with gentle agitation. Remove the solution.

  7. Wash the cells twice in PBS for 1 minute each with gentle agitation.

  8. Prepare a 1:400 dilution of anti-GFP chicken antibody in PBS to obtain a final antibody concentration of 5.0 µg/mL.

  9. Remove the PBS and add the diluted primary antibody solution to the cells. Incubate for 1 hour at room temperature with gentle agitation. Remove the solution.

  10. Wash the cells twice in PBS for 1 minute each with gentle agitation.

  11. Prepare the appropriate conjugated secondary antibody in PBS according to the manufacturer’s recommendations.

  12. Remove the PBS and add the diluted secondary antibody solution to the cells. Incubate for 1 hour at room temperature with gentle agitation. Remove the solution.

  13. Wash the cells twice in PBS for 2 minutes each with gentle agitation. After the final wash, add PBS to the sample. The sample is now ready for imaging and detection using an appropriate method of choice.

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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