Related Technical Notes
This section presents a selected set of primary antibodies for detecting expression tags, including Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP) as well as oligohistidine, hemagglutinin and c-myc. The full range of Invitrogen primary antibodies can be surveyed using our online primary antibody search utility at www.invitrogen.com/handbook/antibodies.
Direct labeling of primary antibodies can be accomplished via amine-reactive chemistry implemented in our comprehensive selection of convenient and easy-to-use protein labeling kits (Kits for Labeling Proteins and Nucleic Acids—Section 1.2). Alternatively, Zenon immunolabeling technology (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3) can be used to prepare fluorophore-, biotin- or enzyme-labeled primary antibodies but is less demanding than chemical labeling methods in terms of antibody quantity and purity requirements.
Expression of the intrinsically fluorescent Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria has become a popular method for following gene expression and protein localization. We offer a rabbit polyclonal antibody that is raised against GFP purified directly from A. victoria. This anti-GFP antibody, which is available as a complete serum (A6455) or as an IgG fraction (A11122) purified by ion-exchange chromatography, facilitates the detection of native GFP, recombinant GFP and GFP fusion proteins by immunofluorescence (), western blot analysis and immunoprecipitation. Several direct conjugates derived from the IgG fraction are also available:
- Green-fluorescent Alexa Fluor 488 rabbit anti-GFP antibody (A21311)
- Orange-fluorescent Alexa Fluor 555 rabbit anti-GFP antibody (A31851)
- Red-fluorescent Alexa Fluor 594 rabbit anti-GFP antibody (A21312)
- Far-red–fluorescent Alexa Fluor 647 rabbit anti-GFP antibody (A31852)
- Biotin-XX rabbit anti-GFP antibody (A10259)
- Horseradish peroxidase (HRP) rabbit anti-GFP antibody (A10260)
In addition, we offer an affinity-purified IgY fraction of chicken anti-GFP antibody, both unlabeled (A10262) and as the biotin-XX conjugate (A10263). Chicken IgY lacks a classic Fc domain and therefore 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 antibodies from both species to be used in a single immunostaining experiment.
Lastly, we offer a rabbit monoclonal anti-GFP antibody (G10362), as well as two mouse monoclonal anti-GFP antibodies. The rabbit monoclonal anti-GFP antibody is raised against full-length GFP and is suitable for applications such as ELISAs, immunohistochemistry, immunocytochemistry, immunoprecipitation and western blotting. Mouse monoclonal 11E5 (anti-GFP, clone 11E5; A11121) is optimized for western analysis, allowing colorimetric detection of as little as 10 ng of GFP or GFP-fusion proteins. Mouse monoclonal 3E6 (anti-GFP, clone 3E6; A11120) is useful for immunoprecipitation, immunocytochemical localization and immunosorbent assays (ELISAs).
We offer an affinity-purified rabbit polyclonal anti–Red Fluorescent Protein antibody (anti-RFP, R10367) for western blot and immunocytochemistry applications. Full-length recombinant denatured and nondenatured TagRFP was used as the immunogen, making the antibody suitable for immunocytochemical detection of the expression products from our CellLight RFP BacMam vectors (CellLight reagents and their targeting sequences—Table 11.1, BacMam Gene Delivery and Expression Technology—Note 11.1).
One common partner in protein fusions is glutathione S-transferase (GST), a protein with natural binding specificity for glutathione that can be exploited to facilitate its purification. We prepare a highly purified rabbit polyclonal anti-GST antibody (A5800) that can be used for western blot analysis, immunodetection and immunoprecipitation of GST fusion proteins. This highly specific and high-titer antibody, which was generated against a 260–amino acid N-terminal fragment of the Schistosoma japonica enzyme expressed in Escherichia coli, is particularly useful for detecting GST distribution in cells. To facilitate the localization of GST and GST-fusion proteins using immunofluorescence techniques, we prepare the anti-GST antibody labeled with our green-fluorescent Alexa Fluor 488 dye (A11131).
Because the GST portion of the fusion protein retains its affinity and selectivity for glutathione, the fusion protein can be conveniently purified from the cell lysate in a single step by affinity chromatography on glutathione agarose (Figure 7.5.1). For the purification of GST fusion proteins, we offer glutathione linked via the sulfur atom to crosslinked beaded agarose, which is available as 10 mL of a sedimented bead suspension (G2879, Detecting Protein Modifications—Section 9.4).
Figure 7.5.1 Coomassie brilliant blue–stained SDS-polyacrylamide gel, demonstrating the purification of a glutathione S-transferase (GST) fusion protein using glutathione agarose (G2879). Lane 1 contains crude supernatant from an Escherichia coli lysate and lane 2 contains the affinity-purified GST fusion protein.
The E. coli β-glucuronidase (GUS) gene (uidA) is a popular reporter gene in plants. For western blot and immunohistochemical analysis of transformed plant tissue and transfected animal cells, we offer unlabeled rabbit anti–β-glucuronidase antibody (A5790) raised against E. coli type X-A β-glucuronidase. Our fluorogenic and chromogenic β-glucuronidase substrates are described in Detecting Glycosidases—Section 10.2.
We offer a polyclonal antibody to the widely used reporter gene product β-galactosidase. Our rabbit anti–β-galactosidase antibody (A11132) is raised against E. coli–derived β-galactosidase and demonstrates high specificity for the enzyme. Whether the enzyme is used as a reporter or as a fusion protein, this antibody provides an easy tool for detecting the β-galactosidase protein. The antibody is suited to a variety of techniques, including immunoblotting, ELISA and immunoprecipitation. Our fluorogenic and chromogenic β-galactosidase substrates are described in Detecting Glycosidases—Section 10.2.
Epitope tagging is a powerful and versatile strategy for detecting and purifying proteins expressed by cloned genes. Protein expression vectors are often engineered with a nucleotide sequence that encodes a peptide epitope tag. Typically, a gene is cloned in-frame relative to the epitope tag such that, upon expression, a fusion protein containing the epitope tag is synthesized. Detection and purification of the epitope-tagged fusion protein can be mediated by antibodies to the engineered peptide, thus eliminating the need for antibodies to proteins from each newly cloned gene. Here we highlight a series of antibodies to common epitopes, including oligohistidine, hemagglutinin (HA) and c-myc, but a more comprehensive list can be found using our online primary antibody search utility at www.invitrogen.com/handbook/antibodies.
The oligohistidine domain is a Ni2+-binding peptide sequence comprising a sequence of four to six histidine residues. When the DNA sequence corresponding to an oligohistidine domain is fused in-frame with a gene of interest, the resulting recombinant fusion protein can be easily purified using a nickel-chelating resin.
In collaboration with QIAGEN, we offer a highly selective antibody to oligohistidine fusion proteins (Penta·His mouse monoclonal antibody, P21315). The antibody is useful for detecting sequences of five or six histidine residues, whether present at the C-terminus, the N-terminus or an internal position in a protein, in both western blots and in immunohistochemistry.
The Penta·His antibody can detect as little as 1–2 ng in western blot applications using fluorescent or chemiluminescent development techniques (); the supplied amount (100 µg) is sufficient for 50–100 minigel blots. This antibody does not recognize tetrahistidine domains or domains in which the histidine string is interrupted by another amino acid. The antibody binds to the oligohistidine domain regardless of the surrounding amino acid context and even when the group is partially hidden, although subtle differences in the amino acid context may change the sensitivity limit for a particular fusion protein. The Penta·His antibody is directly useful for immunoprecipitation, ELISAs and immunohistochemistry.
Our mouse monoclonal anti-hemagglutinin (HA) antibody was raised against the 12–amino acid peptide CYPYDVPDYASL. It recognizes the influenza hemagglutinin epitope YPYDVPDYA, which has been used extensively as a general epitope tag in expression vectors. The extreme specificity of this antibody permits unambiguous identification and quantitative analysis of the tagged protein. This antibody is effective in immunoblotting, immunofluorescence and immunoprecipitation of tagged proteins. We offer Alexa Fluor 488 (A21287) and Alexa Fluor 594 (A21288) conjugates of the anti-HA antibody for those applications that require a directly conjugated antibody.
We offer two antibodies to c-myc, which is commonly used in epitope tagging. Our mouse monoclonal anti–c-myc antibody (IgG1,κ, clone 238-19510; A21280) was raised against the peptide AEEQKLISEEDLLRKRREQLKHKLEQLRNSCA, which corresponds to amino acids 408–439 of the human c-myc protein. The chicken anti–c-myc antibody (IgY, A21281) was raised against the peptide EQKLISEEDL. These antibodies specifically react with the C-terminal epitope (AEEQKLISEEDL) of human c-myc protein encoded in many expression vectors.
For Research Use Only. Not for use in diagnostic procedures.