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Reverse phase protein array for serum and plasma profiling
KTH, School of Biotechnology (BIO).
2014 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Affinity proteomics is often used un biomarker discovery and validation due to the high sample throughput, as compared to for example mass spectrometry approaches. Reverse phase protein arrays (RPPAs) are especially suitable in this regard, since thousands of samples can be analysed simultaneously under identical conditions. RPPA can theoretically be applied to any biological sample, however, tissue and cell lysates are most commonly used, while there are a limited number of studies addressing serum and plasma. This is often explained by the higher sample coplaxity of the latter two, which greatly increases the demand on sensitivity and specificity. The aim of this master thesis project was therefore to investigate the possibilities of applying RPPA to serum and plasma samples, and to optimise the method for the same. Significant differences in the performances of 12 different slide types were observed when comparing parameters such as background intensity, signal-to-noise ratio, binding capacity, spot morphology and positioning, the effect of blocking buffers and washing procedure, dilution curve representation, and sample separation. The different slide types represented various 3-dimensional substrata, e.g. nitrocellulose and hydrogels, and a 2-dimensional epoxide coating. Significant and expected sample separations were seen for various targets, supporting indications of previously suggested limits of detection were seen, and promising reproducibility was observed. UniSart 3D Nitro slides (nitrocellulose substratum) from Sartorius Stedim Biotech demonstrated the most promising results overall and were therefore selected for further method development.

Place, publisher, year, edition, pages
Keyword [en]
Affinity proteomics. protein micro array, plasma profiling, rppa, biomarker discovery
National Category
Engineering and Technology
URN: urn:nbn:se:kth:diva-163677OAI: diva2:801773
Available from: 2015-04-14 Created: 2015-04-10 Last updated: 2015-09-14Bibliographically approved

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