ASGSB 2003 Annual Meeting Abstracts


[79]

antigenic protein from RNALATERTM TREATED CELL CULTURES GROWN ON THE INTERNATIONAL SPACE STATION (ISS), USING THE CELLULAR BIOTECHNOLOGY OPERATIONS SUPPORT SYSTEM (CBOSS).  J.E. Love1, D.K. Hammond2, T.F. Elliott2, K. Holubec2, T.L. Baker2, P.L. Allen3,4 and T.G. Hammond3,41Biological Systems Office, NASA Johnson Space Center, Houston, TX, 2Wyle Life Sciences, Houston, TX, 3V.A. Medical Center, New Orleans, LA and 3Tulane University Health Sciences Center, New Orleans, LA.

   Early CBOSS experiments on the ISS had no means provided to freeze cell culture samples.  Consequently, the samples were treated with RNAlater (Ambion) in order to preserve RNA integrity.  This study was aimed at demonstrating that protein could be recovered after removal of RNA, from cells stored long term in RNAlater.  Initial tests showed protein recovery from cell cultures stored over 3 months in refrigerated RNAlater in amounts equivalent to that from freshly frozen and processed cultures.  Matched flight (ISS Expedition 3) and ground control cell samples from Human Renal Cortical Epithelial (HRCE) cell cultures were compared.  After storage in RNAlater and extraction of RNA (Miniprep, Ambion), the RNA filtrate containing the protein was precipitated, washed and suspended in buffer containing SDS.  Equal concentrations of protein were loaded for each sample and separated by SDS-PAGE and transferred by Western blot to PVDF membrane.  The Western blots were stained with enhanced chemiluminescent ECL Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham Molecular Dynamics).  ImageQuant software was used to quantify the densities of the protein bands.  The ground and flight HRCE cell samples had equivalent amounts of protein, which showed a similar staining pattern over time with antibodies to the Vitamin D receptor and protein kinase C (PKC) beta isoform. 

 

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