ABfinity™ recombinant antibodies for cancer research

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The human body is composed of trillions of cells that grow, divide, and expire in a systematic and temporally regulated manner, as dictated by a complex web of cell signaling pathways. A cell that deviates from this norm can produce atypical or tumorous growth. When left unchecked, a growth irregularity in a single cell can lead to cancer.

Current cancer research is focused on identifying the defects in signaling pathways that eventually lead to aberrations in cell growth, proliferation, differentiation, or senescence. This article describes a set of ABfinity™ recombinant antibodies that target critical molecules in three cell signaling pathways. ABfinity™ antibodies are produced by cloning the heavy and light chains of carefully selected immunoglobulins and then expressing these clones in mammalian cells. Because recombinant antibodies are produced from renewable and stable clones, they provide maximal specificity and sensitivity with every lot.

ABfinity™ antibodies for the p53 signaling pathway

The p53 protein, the product of the tumor suppressor gene TP53, is essential for regulating DNA repair mechanisms, cell cycle, and apoptosis [1,2]. In human tumors, TP53 mutations are more common than mutations in any other gene, underscoring the importance of p53 in the regulation of cell growth. This 53 kDa protein forms a tetrameric transcription factor that binds to specific DNA sequences, where it enhances the transcription rate of cell cycle–arrest genes (including CDKN1A, SFN, TP53I3, and CDC25C) and apoptosis genes (including TNFSF10, BAX, BBC3, and PMAIP1) and represses the transcription of other genes, thereby limiting the propagation of mutations. Through interactions with other binding partners, p53 also plays a regulatory role in angiogenesis, metabolism, and senescence.

More than 30 amino acid residues in the p53 protein serve as targets for posttranslational modifications—including acetylation, methylation, phosphorylation, and ubiquitination—in response to the cell environment and, in particular, stresses that affect chromosomal and DNA integrity. One significant posttranslational modification is acetylation at lysine 382, which is recognized by anti–p53 [AcK382] ABfinity™ recombinant monoclonal and oligoclonal antibodies. Acetylated p53 has been shown to be important for both transcription-dependent and -independent regulation of apoptosis [3]. In research applications, the anti–p53 [AcK382] antibodies have been validated for immunocytochemistry (Figure 1), ELISA, and western blotting (Figure 2A). In addition, we offer anti–p53 [pS15] ABfinity™ monoclonal antibody for detecting p53 phosphorylated at serine 15, which is validated for immunocytochemistry, ELISA, and flow cytometry.

 

anti–p53 [AcK382] ABfinity™ antibodies   Figure 1. Immunocytochemical analysis using anti–p53 [AcK382] ABfinity™ antibodies. HeLa cells were treated with 0.2 µM doxorubicin and 5 mM sodium butyrate for 24 hr and then labeled with either (A) anti–p53 [AcK382] ABfinity™ recombinant rabbit monoclonal antibody or (B) the anti–p53 [AcK382] monoclonal antibody plus 10 µg/mL competing acetylated peptide. Following the primary antibody incubation, both samples were labeled with Alexa Fluor® 488 goat anti–rabbit IgG secondary antibody (green), as well as with the actin stain Alexa Fluor® 594 phalloidin (red) and DAPI nuclear stain (blue). The composite images show nuclear localization of p53 [AcK382]; the near absence of antibody signal in cells treated with both antibody and acetylated peptide demonstrates the antibody’s specificity. Similar results are seen with the anti–p53 [AcK382] ABfinity™ recombinant rabbit oligoclonal antibody.

 

ABfinity™ recombinant antibodies   Figure 2. Western blot analysis using ABfinity™ recombinant antibodies and chemiluminescence-based detection. (A) HeLa cells were treated with 0.2 µM doxorubicin and 5 mM sodium butyrate for 24 hr. Whole cell extracts were analyzed with 2 µg/mL anti–p53 [AcK382] ABfinity™ monoclonal antibody, without (lane 1) or with (lane 2) acetylated peptide competition. (B) Whole cell extracts from HeLa (lane 1) and A431 (lane 2) cells were analyzed with 2.5 µg/mL anti-PRAS40 ABfinity™ monoclonal antibody. (C) Whole cell extracts from Colo 205 cells were analyzed with 2 µg/mL anti–Rb [pT821] ABfinity™ monoclonal antibody, without (lane 1) or with (lane 2) phosphopeptide competition. Similar results are seen when using the corresponding ABfinity™ oligoclonal antibodies in western blot analysis.

ABfinity™ antibodies for the Akt/mTOR signaling pathway

The Akt/mTOR pathway is a broadly influential signaling network that sits just downstream of the phosphatidylinositol 3-kinase (PI3K) pathway. Because of its regulatory roles in cell growth and proliferation, metabolism, and apoptosis, dysregulation of the Akt/mTOR pathway is one of the hallmarks of many cancers. The 40 kDa proline-rich Akt substrate (PRAS40) appears to function at a critical junction between Akt and mTORC1, acting as both a substrate and an insulin-regulated inhibitor of the mTORC1 kinase [4–6]. PRAS40 silencing inactivates the Akt/mTOR pathway, suggesting PRAS40 as a potential target for cancer therapies.

We offer ABfinity™ recombinant monoclonal and oligoclonal antibodies that recognize PRAS40, as well as antibodies that specifically recognize PRAS40 phosphorylated at threonine 246. These anti-PRAS40 ABfinity™ antibodies have been validated for ELISA and western blotting (Figure 2B) in research applications.

ABfinity™ antibodies for the Rb/E2F signaling pathway

The retinoblastoma (Rb) protein is a tumor suppressor protein that regulates cell proliferation and apoptosis through the Rb/E2F signaling pathway. Hyperphosphorylated Rb enhances cell proliferation, whereas hypophosphorylated Rb inhibits cell proliferation and prevents the replication of damaged DNA. Specifically, dephosphorylated Rb negatively regulates the G1/S transition by binding and inactivating the E2F transcription factors [7]. Dephosphorylation of Rb at threonine 821 is also required for apoptosis induced by UV and by the inhibition of cyclin-dependent kinases (Cdk) [8].

We offer phosphorylation site–specific ABfinity™ recombinant monoclonal and oligoclonal antibodies that recognize Rb [pT821] and Rb [pS780], and these antibodies are validated for immunocytochemistry (Figure 3), ELISA, and western blotting (Figure 2C) in research applications. Also available is anti-Rb [pSpT249/252] ABfinity™ oligoclonal antibody, which can be used for ELISA and western blotting.

 

anti–Rb [pT821] ABfinity™ antibodies   Figure 3. Immunocytochemical analysis using anti–Rb [pT821] ABfinity™ antibodies. U2OS cells were labeled with either (A) anti–Rb [pT821] ABfinity™ recombinant rabbit oligoclonal antibody or (B) anti–Rb [pT821] plus a competing Rb phosphopeptide. Following the primary antibody incubation, both samples were labeled with Alexa Fluor® 488 goat anti–rabbit IgG secondary antibody (green), as well as with the actin stain Alexa Fluor® 594 phalloidin (red) and DAPI nuclear stain (blue). The composite images show nuclear localization of Rb [pT821]; the lack of antibody signal in cells treated with both antibody and phosphopeptide demonstrates the antibody’s specificity. Similar results are seen with the anti–Rb [pT821] ABfinity™ recombinant rabbit monoclonal antibody.

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