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.

Antibodies Specific for Reporter Gene Products

Anti–Green Fluorescent Protein (GFP) Antibodies

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.ref 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 ref (photo), western blot analysis ref 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).


Anti–Red Fluorescent Protein (RFP) Antibody

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).


Anti–Glutathione S-Transferase Antibody

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.ref 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.ref 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.ref 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 ref (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).

 

polyacrylamide gel 

 

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.

 

Anti–β-Glucuronidase Antibody

The E. coli β-glucuronidase (GUS) gene (uidA) is a popular reporter gene in plants.ref For western blot and immunohistochemical analysis of transformed plant tissue ref and transfected animal cells,ref 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.


Anti–β-Galactosidase Antibody

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.

Antibodies Specific for Epitope Tags

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.


Penta·His Antibody

The oligohistidine domain is a Ni2+-binding peptide sequence comprising a sequence of four to six histidine residues.ref 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.ref

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 (photo); 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.


Anti-HA Antibody

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.ref 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.


Anti–c-myc Antibody

We offer two antibodies to c-myc, which is commonly used in epitope tagging.ref 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) ref of human c-myc protein encoded in many expression vectors.

Ordering Information

Sku Name Size Price Qty
A11133 BSA (Bovine Serum Albumin) Rabbit pAb 0.5 mL USD 145.00
A6428 CPY Mouse Monoclonal Antibody (clone 10A5B5) 250 µg USD 390.00
A21281 c-Myc Chicken IgY Antibody Fraction 100 µL USD 393.00
A21280 c-Myc Mouse IgG1 Monoclonal Antibody (clone 289-19510) 100 µL USD 381.00
A6429 DMP1 yeast Mouse Monoclonal Antibody (clone 5C5A7) 250 µg USD 390.00
A11132 β-Galactosidase, rabbit IgG antibody fraction 0.5 mL USD 127.00
A21282 GFAP Monoclonal Antibody, Mouse (131-17719) 100 µL USD 368.00
A21294 GFAP Mouse Monoclonal Antibody (clone 131-17719), Alexa Fluor® 488 Conjugate 50 µL USD 340.00
A21295 GFAP Mouse Monoclonal Antibody (clone 131-17719), Alexa Fluor® 594 Conjugate 50 µL USD 368.00
A5790 β-Glucuronidase, rabbit IgG antibody fraction 0.5 mL USD 290.00
A5800 Glutathione S-transferase Rabbit IgG Antibody Fraction 0.5 mL USD 293.00
A11131 Glutathione S-transferase, Rabbit IgG Antibody Fraction, Alexa Fluor® 488 Conjugate 0.5 mL USD 289.00
A21270 Golgin-97 Monoclonal Antibody, Mouse (CDF4) 100 µg USD 220.00
A10262 GFP Chicken IgY Antibody Fraction 100 µL USD 297.00
A10263 GFP Chicken IgY Antibody Fraction, Biotin-XX Conjugate 100 µL USD 297.00
A11120 GFP (clone 3E6) Mouse IgG2a, Monoclonal Antibody 100 µg USD 246.00
A11121 GFP (clone 11E5) Mouse IgG1, Monoclonal Antibody 100 µg USD 246.00
A11122 GFP Rabbit IgG Polyclonal Antibody Fraction 100 µL USD 310.00
A21311 GFP Rabbit IgG Antibody Fraction, Alexa Fluor® 488 Conjugate 100 µL USD 310.00
A31851 GFP Rabbit IgG Antibody Fraction, Alexa Fluor® 555 Conjugate 100 µL USD 316.00
A21312 GFP Rabbit IgG Antibody Fraction, Alexa Fluor® 594 Conjugate 100 µL USD 310.00
A31852 GFP Rabbit IgG Antibody Fraction, Alexa Fluor® 647 Conjugate 100 µL USD 207.00
A10259 GFP Rabbit IgG Antibody Fraction, Biotin-XX Conjugate 100 µL USD 297.00
A10260 GFP Rabbit IgG Antibody Fraction, HRP Conjugate 200 µg USD 297.00
A6455 GFP Rabbit Serum Polyclonal Antibody 100 µL USD 207.00
A6427 V-ATPase 60 kDa subunit Mouse Monoclonal Antibody (clone 13D11B2) 250 µg USD 390.00
A21287 HA (clone 16B12) Mouse IgG1 Monoclonal Antibody, Alexa Fluor® 488 Conjugate 100 µL USD 384.00
A21288 HA (clone 16B12) Mouse IgG1 Monoclonal Antibody, Alexa Fluor® 594 Conjugate 100 µL USD 394.00
A21271 HuC/HuD Neuronal Protein Mouse Monoclonal Antibody (clone 16A11) 100 µg USD 368.00
A21272 HuC/HuD Neuronal Protein Monoclonal Antibody, Mouse (16A11) Biotin-XX 100 µg USD 435.00
A21277 HuR mRNA-Binding Protein Mouse anti-Human Monoclonal Antibody (clone 19F12) 100 µg USD 368.00
A6473 NMDA Receptor Subunit 2A Rabbit anti-Rat Polyclonal Antibody 10 µg USD 410.00
A6474 NMDA Receptor Subunit 2B Rabbit anti-Rat Polyclonal Antibody 10 µg USD 410.00
A21359 Complex I Subunit NDUFB6 Mouse Monoclonal Antibody (clone 21C11BC11) 100 µg USD 368.00
A6404 Complex IV Subunit II Mouse Monoclonal Antibody (clone 12C4F12) 100 µg USD 368.00
A21347 Complex IV Subunit IV Mouse Monoclonal Antibody (clone 10G8D12C12) 100 µg USD 368.00
A21348 Complex IV Subunit IV Mouse Monoclonal Antibody (clone 20E8C12) 100 µL USD 368.00
A21355 ATP Synthase Subunit IF1 Mouse mAb (clone 5E2D7) 100 µg USD 368.00
A21351 ATP Synthase Subunit Beta Mouse mAb (clone 3D5AB1) 100 µg USD 368.00
A21273 Pep12p (yeast) Mouse Monoclonal Antibody (clone 2C3G4) 50 µg USD 390.00
A21327 Phosphatidylinositol 4,5-Diphosphate Mouse IgM, Monoclonal Antibody (clone 2C11) 100 µL USD 365.00
A21328 Phosphatidylinositol 3,4,5-Triphosphate Mouse IgM Monoclonal Antibody (clone RC6F8) 100 µL USD 365.00
A21326 PDH subunit E2/E3bp Mouse Monoclonal Antibody (clone 13G2AE2BH5) 100 µg USD 368.00
A6442 Synapsin I Rabbit anti-Bovine Polyclonal Antibody 10 µg USD 240.00
A11130 Transferrin Receptor Monoclonal Antibody, Mouse (clone 236-15375) 50 µg USD 200.00
A11126 Bovine Alpha-Tubulin Mouse mAb (clone 236-10501) 50 µg USD 200.00
A21371 Alpha-Tubulin Mouse anti-Bovine mAb (clone 236-10501), biotin conjugate 50 µg USD 260.00
A23210 APO-BrdU™ TUNEL Assay Kit, with Alexa Fluor® 488 Anti-BrdU 60 assays USD 524.00
Q10013 CD14 Mouse Anti-Human mAb (clone TÜK4), Qdot® 605 conjugate 100 µL USD 499.00
Q10056 CD14 Mouse Anti-Human mAb (clone TüK4), Qdot® 655 conjugate 100 µL USD 499.00
Q10064 CD14 Mouse Anti-Human mAb (clone TüK4), Qdot® 800 conjugate 100 µL USD 506.00
Q10065 CD27 Mouse Anti-Human mAb (clone CLB-27/1), Qdot® 605 conjugate 100 µL USD 499.00
Q10066 CD27 Mouse Anti-Human mAb (clone CLB-27/1), Qdot® 655 conjugate 100 µL USD 499.00
Q10012 CD3 Mouse Anti-Human mAb (clone S4.1 (AKA 7D6)), Qdot® 655 conjugate 100 µL USD 499.00
Q10053 CD38 Mouse Anti-Human mAb (clone HIT2), Qdot® 605 conjugate 100 µL USD 499.00
Q10051 CD45 Mouse Anti-Human mAb (clone HI30), Qdot® 605 Conjugate 100 µL USD 499.00
Q10062 CD45 Mouse Anti-Human mAb (clone HI30), Qdot® 705 Conjugate 100 µL USD 512.00
Q10060 CD4 Mouse Anti-Human mAb (clone S3.5), Qdot® 705 Conjugate 100 µL USD 506.00
Q10008 CD4 Mouse Anti-Human mAb (clone S3.5), Qdot® 605 Conjugate 100 µL USD 499.00
Q10007 CD4 Mouse Anti-Human mAb (clone S3.5), Qdot® 655 Conjugate 100 µL USD 499.00
Q10009 CD8 Mouse Anti-Human mAb (clone 3B5), Qdot® 605 Conjugate 100 µL USD 499.00
Q10055 CD8 Mouse Anti-Human mAb (clone 3B5), Qdot® 655 Conjugate 100 µL USD 499.00
Q10059 CD8 Mouse Anti-Human mAb (clone 3B5), Qdot® 705 Conjugate 100 µL USD 506.00
Q10156 CD45 Mouse Anti-Human mAb (clone H130), Qdot® 800 Conjugate 100 µL USD 510.00
Q10153 CD10 Mouse Anti-Human mAb (clone MEM-78), Qdot® 605 conjugate 100 µL USD 499.00
Q10172 CD2 Mouse Anti-Human mAb (clone S5.5), Qdot® 605 conjugate 100 µL USD 499.00
Q10179 CD19 Mouse Anti-Human mAb (clone SJ25-C1), Qdot® 655 conjugate 100 µL USD 499.00
Q10069 CD45RA Mouse Anti-Human mAb (clone MEM-56), Qdot® 655 Conjugate 100 µL USD 499.00
Q10047 CD45RA Mouse Anti-Human mAb (clone MEM-56), Qdot® 605 Conjugate 100 µL USD 499.00
Q10092 CD4 Rat Anti-Mouse mAb (clone RM4-5), Qdot® 605 Conjugate 100 µL USD 399.00
Q10176 CD45R (B220) Rat Anti-Mouse mAb (clone RA3-6B2), Qdot® 655 Conjugate 100 µL USD 385.00
Q10054 CD3 Mouse Anti-Human mAb (clone UCHT1), Qdot® 605 conjugate 100 µL USD 499.00
Q10052 HLADR (class II) Mouse Anti-Human mAb (clone Tü36), Qdot® 605 Conjugate 100 µL USD 499.00
Q10063 HLADR (class II) Mouse Anti-Human mAb (clone Tü36), Qdot® 800 Conjugate 100 µL USD 506.00
Q10076 Mouse IgG2a, Qdot® 705 conjugate 100 µL USD 240.00
Q10014 Mouse IgG2a, Qdot® 605 conjugate 100 µL USD 233.00
Q10015 Mouse IgG2a, Qdot® 655 conjugate 100 µL USD 233.00
P21315 Penta•His™ Mouse IgG1, Monoclonal Antibody (clone Anti-Pentahistidine), BSA-free 100 µg USD 626.00
S34200 SelectFX® Alexa Fluor® 488 Endoplasmic Reticulum Labeling Kit, for fixed cells 1 kit USD 504.00
S34201 SelectFX® Alexa Fluor® 488 Peroxisome Labeling Kit, for fixed cells 1 kit USD 504.00
For Research Use Only. Not for use in diagnostic procedures.