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Survivin, an inhibitor of apoptosis protein (IAP) is a pro-survival molecule that is increased in nearly every human tumor studied. In head and neck squamous cell carcinoma, Survivin levels are significantly greater than in normal upper aerodigestive mucosa. High Survivin levels in these tissues correlate with a higher probability of nodal metastasis and loco-regional recurrence, but may also indicate higher radiosensitivity. Survivin includes multiple sites for post-translational modification, including 4-5 major phosphorylation sites. |
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In this era of targeted therapeutics, there is a need for highly sensitive and quantitative methods to measure proteins in clinical specimens to define and predict of specific therapies for patients. However, the current gold standard in proteomic analysis, Western Immuno Blotting, has many manual steps, is insensitive and provides only semi-quantitative data. Here, we present the first report of a novel automated instrument “Sally™,” a nanovolume size-based protein separation platform used to quantify proteomic profiles of clinical specimens. Since the instrument automates all steps of proteomic analysis including sample loading, size-based protein separation, immunoprobing, washing, detection and data quantification, we are able to make up to 96 measurements in a single experiment, in a highly quantitative manner, minimizing errors due to manual variability. We were able to quantify proteins in either surgical specimens preserved at -80C in Optimum Cutting Temperature Compound (OCT) or fine needle aspirates flash-frozen at time of collection. Tissues were homogenized and lysed in a commercially available Bicine/CHAPS lysis buffer, denatured and loaded in duplicate in a 384-well plate at a final concentration of 0.2-2 mg/ml in each well. 40 nanoliters of lysate was used for each protein measurement. We measured 10 proteins (including AKT and ERK) from the MAPK signaling pathways, normalized to loading controls (β-actin or tubulin). It took 1 hour to prepare a run and 14 hours of unattended automated machine time to generate the analyzed data. Therefore this technology enables us to report highly quantified protein profiles to the clinical team in less than 24 hours of receiving a clinical sample. In the course of 1 week, we quantified ERK1 and ERK2 in over 100 clinical specimens and in a subset of 22, measured an additional panel of proteins including PI3K proteins [AKT1, AKT2, pan-AKT, GSK3b, S6]. The ability to make rapid and quantitative measurements will be useful to measure and predict responses to targeted therapeutics. The Sally platform is an extremely efficient, quantitative and high-throughput platform that can be used to rapidly generate proteomic profiles for clinical specimens for the development of novel diagnostic and predictive biomarkers. |
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Aberrant expression and signaling of multiple proteins in the NF-κB pathway are commonly associated with inflammatory and stress-induced diseases, including many cancers. Understanding how NF-κB signaling impacts disease progression is important to the development of novel therapeutics. Cell signaling events are routinely assessed using traditional Western blot analysis. The Western blot technique is very labor intensive and generally yields results that are semi-quantitative. The Simple Western platform described here completely automates the manual steps involved in traditional Western blot protocols and can analyze up to 96 samples in a single experiment. Because Simple Western protocols consume only microliter sample volumes, reproducible and quantitative results can be generated from precious or quantity-limited samples.
We present, for the first time, results generated on Sally, the newest member of the Simple Western platform from ProteinSimple. Sally is easy to set-up and runs hands-free up to 96 data points in a single experiment, thus addressing the need for higher throughput. Sally generates 8 individual measures from 5 µL of sample which allows for characterization of whole signaling pathways from one small sample size.
Data generated on Sally from examination of targets in the NF-κB (p100/p50) pathway demonstrate high reproducibility and low intra-assay variability. Response to TNF-α treatment in whole cell and nuclear extracts of IκB and NF-κB subunits (c-Rel, p65, and p50/p105) demonstrates, as expected, statistically significant changes in signal and localization. Results and workflow comparisons indicate a distinct advantage of the Simple Western when compared to traditional Western methods. |
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