ASGSB 2003 Annual Meeting Abstracts
antigenic protein from
RNALATERTM TREATED CELL CULTURES GROWN ON THE INTERNATIONAL
SPACE STATION (ISS), USING THE CELLULAR BIOTECHNOLOGY OPERATIONS SUPPORT
J.E. Love1, D.K. Hammond2, T.F. Elliott2, K.
Holubec2, T.L. Baker2, P.L. Allen3,4 and T.G.
Hammond3,4. 1Biological 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 RNAlaterÒ
(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 RNAlaterÒ.
Initial tests showed protein recovery from cell cultures stored over 3 months
in refrigerated RNAlaterÒ
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
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 ECLÔ
Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer
(Amersham Molecular Dynamics). ImageQuantÒ
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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