I am hoping to collect a secretory protein from fresh and flash frozen breast tissue for analysis by proximity ligation assay. Specifically, I am in need of a protocol which would allow me to wash a sample of whole tissue and only analyze the wash, thereby avoiding any unnecessary damage to the tissue sample.
I have not worked with human tissue but I have collected secreted proteins from a fungal agar culture without lysing mycelia by washing the agar with KH2PO4.
This protocol was adapted from a combination of two protocols: Imanaka et al. (2010) and Fragner et al. (2009) . Cultures grown on an agar medium were ground gently using a pestle and mortar to break up the agar but not to grind mycelia. KH2PO4 (50 mM, 50 ml) was added gradually to a 25 ml agar culture. This was agitated overnight at 4 °C. The liquid was gravity filtered before being centrifuged at 48,000 g (4 °C) for 30 min and supernatants were put into clean containers. 100 % (w/v) TCA stock solution (Section 2.1.5.15) was added to the liquid so that it comprised 10 % (v/v) of the final solution. Samples were mixed and incubated on a roller overnight at 4 °C, followed by centrifugation at 1700 g (4 °C), 45 min. Tris-buffer (Section 2.1.5.3), (50 mM, 2 ml) was added to ice cold acetone (final concentration of Tris-buffer 20 % (v/v)). This solution (1 -2 ml) was used to wash the pellet. This was centrifuged for 2 min at 9,400 g (4 °C) and washed twice more with centrifugation for 1 min at 9,400 g (4 °C), after which it was washed with 1 ml of ice-cold acetone; air dried and stored at −20 °C.
Fragner, D., Ku, U., Zomorrodi, M., Kues, U., and Majcherczyk, A. (2009). Optimized protocol for the 2-DE of extracellular proteins from higher basidiomycetes inhabiting lignocellulose. Electrophoresis 30: 2431–2441.
Imanaka, H., Tanaka, S., Feng, B., Imamura, K., and Nakanishi, K. (2010). Cultivation characteristics and gene expression profiles of Aspergillus oryzae by membrane-surface liquid culture, shaking-flask culture, and agar-plate culture. Journal of Bioscience and Bioengineering 109: 267–273.
Thank you for the answer Cassandra,
This is a uniquely challenging position as I cannot utilize a cell culture. The goal for my project is to investigate alternative markers for biospecimen quality, and as such, I am interested in using whole, flash frozen tissue in a manner that does not require any unneccesary damage to the tissue (such as homogenation and lysis). Therefore, I am in search of a protocol which requires only thawing the tissue, followed by a wash of the tissue in an appropriate buffer sollution. Only the tissue wash would be analyzed for protein content. I am aware that this method would yield extremely minute amounts, if any, of the secreted protein of interest, however, I hope that the sensitivity of proximity ligation assays may allow for the quantification of such miniscule concentrations.
You have my gratitude for the information you provided Florian. That seems to be the most promising avenue I have yet seen for the information I hope to gather.
My only experience in collecting secretory proteins from intact cells includes a step to ensure that the proteins are massively secreted from secretory granules... we use dibucaine for Tetrahymena to do this clearly the stimulus should be tissue appropriate. Opening Ca2+ channels perhaps? Florian's separation protocol sounds perfect.
Thank you for the input Anne, however I am trying to avoid as much manipulation of the tissue as possible. We want any downstream assays for gene expression or proteome investigation to remain as much unaltered as possible by the quality testing method.
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