Proteintech Antibodies and Near-infrared Imaging Technologies
Proteintech is always searching to validate new and interesting ways you can put our high-quality antibodies to use. Sometimes, inspiration comes from the work you are already undertaking with our antibodies. This week, we have been reviewing a growing number of publications by authors using both Proteintech antibodies and the Odyssey Infrared Imaging System, developed by LI-COR Biosciences. The best examples of these data for your perusal are hand-picked. These examples not only represent work from several areas of life science research – including neuroscience, cancer research and virology – they also represent a wealth of data, validating Proteintech antibodies for use in tandem with LI-COR’s™ Odyssey system. Importantly, this offers you an alternative to traditional chemiluminescence methods for the detection of proteins with our antibodies via Western blot (WB). The writing here may even serve to enlighten those who are unfamiliar with infrared imaging systems or those who are (ironically) stuck in the dark ages of chemiluminescence.
Near-Infrared fluorescence versus chemiluminescence
Firstly, what advantages does near-infrared (NIR) fluorescence have over more traditional chemiluminescence detection methods? Well, firstly, there are no dark rooms or messy equipment required; developer and fixer stains are never a great look in a neat and tidy lab! Aesthetics and ease of use aside, where NIR-fluorescence technologies really have the edge is their imaging superiority over chemiluminescence. The figure provided below is a perfect demonstration of this. NIR fluorescence detection can achieve such sensitivity and low background because it does not rely on the visible wavelength range. In this range, membranes and plastics used in WB imaging produce a high background signal, limiting the sensitivity of chemiluminescence. NIR fluorescence avoids this problem as both autofluorescence and light scatter are dramatically reduced at the NIR wavelengths.
So, you now know a little of the theory as to why NIR fluorescence is a favorable alternative for detecting Proteintech antibodies, but how is this technology being used out there, in research featuring our antibodies?
TARDBP and Wobbler mice: An example of two-color detection and quantification
The TARDBP gene encodes TAR DNA-binding protein 43 (TDP-43), and our rabbit polyclonal antibody against this protein (10782-2-AP) has featured in over 200 publications. These publications investigate and report on TDP-43’s role in many neuropathologies, including the progressive motor neuron disease Amyotrophic lateral sclerosis (ALS); a disease which can be either hereditary (familial ALS – fALS) or sporadic.
A 2009 Neuroscience paper, by authors Dennis and Citron (PMID: 19013502), comments on the resurgence of Wobbler mice in the research of motor neuron disease and, in particular, ALS. Previously, Wobbler mice had been employed widely in the study of ALS, but their usage in neuroscience research began to decline after the development of the SOD1 transgenic mouse strain. This strain carries the human fALS mutations, and at first was thought to be a more representative model of all forms of ALS than Wobbler. However, the SOD1 mice have since been shown to lack the TDP-43 changes associated with sporadic ALS. What Dennis and Citron were able to confirm, using our TDP-43 antibody and the LI-COR Oddyssey system, was that Wobbler mice mimicked the delocalization of elevated TDP-43 protein seen in sporadic ALS, characterized by its aberrant cytoplasmic localization. Using these technologies, they were able to easily and reliably visualize and quantify western bands representing TDP-43 amounts in the cytoplasm and nucleus of both Wobbler and control mice (See figure and legend below, extracted, with permission, from Dennis and Citron, (2009) Neuroscience). Moreover, they were able to visualize GAPDH simultaneously and use its signal to standardize TDP-43 quantification. This is a simple yet elegant example of where two-colour detection and quantification has been used.
NIR fluorescence technology for standard WB imaging of TFF3 and ARG2: Discovering biomarkers of metastasis.
- Human ileum were subjected to SDS PAGE followed by western blot with 11810-1-AP(TFF3 Antibody) at dilution of 1:1500
Metastasis to the lymph node is a major cause for concern in any type of cancer; lymph nodes in the local vicinity of primary cancers are routinely biopsied during curative surgery to check for metastatic events. Colorectal cancer (CRC) is no exception to this and is one of the leading causes of cancer-related death worldwide. In 2008, the American Association of Cancer Research (AACR) reported that there were 150,000 new cases of CRC and over 50,000 CRC-related deaths in the US. The need for biomarkers identifying colorectal cancer development and predicting its metastasis is pressing.
Cancer proteomics has been increasingly important in cancer biomarker discovery and the efforts of scientists in this field have turned to uncovering the cancer “secretome”, i.e. the full array of proteins secreted into plasma or surrounding fluids. It is hoped that identifying the secretome of cancers and comparing them with those of healthy tissues will uncover potential diagnostic and predictive markers. In the case of metastasis prediction, the secretomes of primary and secondary cancers should be compared. This is exactly what Xue et al. did in their 2009 Journal of Proteome Research paper (PMID: 19924834). They used the LI-COR Odyssey system and the Proteintech antibodies against TFF3 and ARG2 to help identify six secreted proteins unique to a lymph node metastatic cell line (SW620) compared to its originating primary CRC cell line, (SW480) from the same patient. TFF3 was identified as a strong candidate for the prediction of CRC lymph node metastasis. There are also some beautiful examples of immunohistochemistry visulisation of TFF3 expression in both healthy and CRC tissue in this publication. You can view this publication here.
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- Human stomach were subjected to SDS PAGE followed by western blot with 12275-1-AP(AGR2 Antibody) at dilution of 1:2000
- Immunohistochemistry of paraffin-embedded Breast cancer using 12275-1-AP(AGR2 Antibody) at Dilution 1:100 (under 10x lens)
- Hela cell were subjected to SDS PAGE followed by western blot with 11714-1-AP(IFITM3 Antibody) at dilution of 1:2000
- Immunohistochemistry of paraffin-embedded Lung cancer using 11714-1-AP(IFITM3 Antibody) at Dilution 1:100 (under 10x lens)
IFTM2 and IFITM3 used to validate in-cell Western assay analysed using the LI-COR odyssey system:
Discovery of cellular proteins that inhibit West-Nile virus and Dengue virus
West-Nile virus (WNV) and Dengue virus (DENV) are mosquito borne flaviviruses that cause invasive neurological disease and fatal hemorrhagic fever in humans. Type I Interferons are key mediators of the host innate antiviral immune response, triggering the expression of interferon-stimulated genes (ISGs) for this process. A paper published in mid-2010, in the Journal of Virology (PMID: 20534863), identifies five such ISGs that efficiently suppress WNV or DENV when expressed in HEK293 cells prior to infection. Of these IFITM2 and IFITM3 were observed to disrupt early steps (viral entry/uncoating) of viral infection by In-Cell Western assay. This technology, pioneered by LI-COR, enabled the authors to detect viral envelop protein whilst simultaneously monitoring cell viability with Sapphire 700 (LI-COR); this allowed normalization of the data. Our IFITM3 antibody (11714-1-AP) was used to further validate the above data in the antiviral response to DENV. WB detection of IFITM3 in HeLa cells incubated with IFITM3-specific or control siRNA, confirmed IFITM3’s knockdown. This enabled the authors to reach the conclusion that diminished antiviral response to infection with DENV was due to depletion of IFITM3 at the mRNA and protein levels in these cells. The paper also used our MxA antibody (13750-1-AP), however MxA showed no change in expression upon activation of the type I Interferon signal transduction pathway in response to WNV or DENV. If you have access to publications on the Journal of Virology website, you can download this paper here.
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- Hela cell were subjected to SDS PAGE followed by western blot with 11714-1-AP(IFITM3 Antibody) at dilution of 1:2000
- Immunohistochemistry of paraffin-embedded Lung cancer using 11714-1-AP(IFITM3 Antibody) at Dilution 1:100 (under 10x lens)
- MCF7 cell were subjected to SDS PAGE followed by western blot with 12769-1-AP(IFITM2 Antibody) at dilution of 1:2000
- Immunohistochemistry of paraffin-embedded Ovary cancer using 12769-1-AP(IFITM2 Antibody) at Dilution 1:100 (under 10x lens)
- Mouse spleen were subjected to SDS PAGE followed by western blot with 13750-1-AP(MX1 Antibody) at dilution of 1:1200
