HeLa cells were treated with 60 µM chloroquine overnight. The following day, cells were fixed and permeabilized and subsequently labeled with 1 µg/mL anti-LC3B with Invitrogen™ Goat anti-Rabbit Secondary Antibody, Alexa Fluor™ 488 green (Cat. No. A-11034). Cells were counterstained with Invitrogen™ HCS Cellmask™ Deep Red (purple) and 1 µg/mL Hoechst 33342 (blue) stains. Cells were imaged on a Nikon D200 wide-field microscope.
Anti-Rabbit Secondary Antibodies
Our anti-rabbit secondary antibodies are affinity-purified polyclonal or monoclonal antibodies with well-characterized specificity for rabbit immunoglobulin classes and fragments. They are available in host species including goat, donkey, mouse, chicken, and sheep. Our anti-rabbit secondary antibodies also come in a variety of conjugates:
- Unlabeled antibodies
- Enzyme conjugates (alkaline phosphatase (AP) or horseradish peroxidase (HRP))
- Fluorescent conjugates (Alexa Fluor, Fluorescein (FITC), and other classic fluorescent conjugates
Anti-rabbit secondary antibodies are generated by immunizing a host animal with a pooled population of immunoglobulins (Ig) from the target species. In this case, the Ig would come from rabbits and be introduced into one of the available host species: goat, donkey, mouse, chicken, or sheep. After the host animal’s immune system has responded to the rabbit Ig, the resulting antibodies are collected and purified.
Our anti-rabbit secondary antibodies are available as whole IgG or the F(ab')2 fragment. Each of these antibodies is affinity purified using its target antigen (the primary antibody class and fragment) coupled to an agarose support. This affinity purification helps eliminate cross-reactivity from nonspecific proteins, resulting in high specificity and low background signal. Conjugated antibodies are affinity purified before labeling.
Selected secondary antibodies have been further purified by adsorbing the antigen-purified antibody to an affinity column containing immobilized serum proteins or IgG of selected species. This additional processing minimizes antibody cross-reactivity to the serum proteins of those selected species. These antibodies are designated as cross-adsorbed or highly cross-adsorbed, based on the amount of processing that has occurred.
Featured Product Data
Immunohistochemistry using Invitrogen™ NMDA Receptor Subunit 2A Polyclonal Antibody, Rabbit: rat brain cryosections labeled with anti–NMDA receptor, subunit 2A (rat) antibody, rabbit IgG fraction (Cat. No. A6473) and detected using Invitrogen™ Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor™ 488 conjugate (Cat. No. A11008). The tissue was also labeled with Invitrogen™ GFAP / Glial Fibrillary Acid Protein Antibody, Alexa Fluor™ 594 conjugate (Cat. No. A21295) and counterstained with Invitrogen™ TOTO™-3 iodide (Cat. No. T3604), which was pseudocolored light blue in this image.
Immunofluorescence analysis of neurons using Invitrogen™ anti-beta III tubulin antibody (Cat. No. 480011). GABAergic precursor cells derived from H9 embryonic stem cells were differentiated for 7 days and stained with antibodies against GABA (Cat. No. PA5-32241 at 1:5,000) followed by Invitrogen™ Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor™ 488 conjugate (Cat. No. A21206, green) and neuronal marker beta III tubulin (Cat. No. 480011 at 1:1,000) followed by Invitrogen™ Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor™ 594 conjugate (Cat. No. A11005, red). Nuclear DNA was stained with DAPI (blue).
How to find a secondary antibody
Step 1: Search for the target species of interest (e.g., “anti-mouse” or “goat anti-rabbit” or “alexa fluor 488”).
Step 2: Narrow results by host species, conjugate, application, and other criteria using side filters.
Annotated Product References
Goat anti-Rabbit IgG (H+L) Secondary Antibody, Polyclonal (A-11034) was used in immunocytochemistry to discuss the limitations of using microtubule-associated protein 1 light chain 3 (LC3) and p62 to assess autophagy. Gómez-Sánchez R, Pizarro-Estrella E, Yakhine-Diop SM et al. (2015) Routine western blot to check autophagic flux: cautions and recommendations. Analytical Biochemistry 477: 13-20.
Goat anti-Rabbit IgG (H+L) Secondary Antibody, Polyclonal (A-11008) was used in immunohistochemistry - frozen section to use small hairpin RNAs to specifically knock down alpha 3–containing sodium pumps in different regions of the adult mouse brain. Fremont R, Tewari A, Khodakhah K. (2015) Aberrant Purkinje cell activity is the cause of dystonia in a shRNA-based mouse model of rapid onset dystonia-parkinsonism. Neurobiology of Disease 82: 200-212.
Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Polyclonal (A-21206) was used in immunocytochemistry to show that Basonuclin-1 regulates TGF-β-induced epithelial dedifferentiation of mammary epithelial cells. Feuerborn A, Mathow D, Srivastava PK et al. Basonuclin-1 modulates epithelial plasticity and TGF-β1-induced loss of epithelial cell integrity. Oncogene 34(9): 1185-1195.
Thermo Scientific™ Superclonal™ secondary antibodies represent a breakthrough in recombinant antibody technology designed to provide precise and accurate detection of mouse, rabbit and goat primary antibodies in a variety of applications. Each Superclonal secondary antibody is formulated and optimized to help achieve excellent results in ELISA, western blot, and cell imaging.
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