ABSTRACT
A good loading control is critical for accurate comparison of tissue protein levels in aged and young tissue. After reviewing the literature we discovered that the housekeeping proteins commonly used in Western analysis had significant drawbacks. The purpose of this study was to evaluate the linearity and reproducibility of common housekeeping proteins tubulin, actin and GAPDH compared to measures of total protein staining using Direct Blue and Stain Free gels. We found that measurements of total protein staining were superior to housekeeping proteins both in linearity and reproducibility in young and old mouse, liver, heart, and brain. Among the total protein staining methods, the Stain Free method was superior to Direct Blue, in terms of lower variability and higher accuracy in all tissues, at both ages.
ABSTRACT
Cross-talking between heat shock proteins (HSPs) and cold inducible proteins (CIPs) subsequent to combinational mild heat (35°C) and cold (8°C) stress was investigated in vivo for four cultivars of Solanum tuberosum L. viz. Kufri Pukhraj (PO), Kufri Jyoti (GS), Kufri Ashoka (KF) and Kufri Chandramukhi (CM) in the order of their decreasing thermotolerance, to understand how this economic crop adapts to extreme temperature fluctuation. We showed a time-course differential genotypic expression pattern for HSPs at 35°C for 10h and CIPs at 8°C for 12h time-lapse. Remarkably, we noted the disappearance of a housekeeping protein (HKP) of about 19.8KD at 2h, 35°C in GS absent in CM, KF and PO; but strongly expressed as CIPs at 8°C for all the cultivars. Furthermore, heat-stress led to an outstanding transient induction of HSP95.9, HSP83.6, HSP78.7, HSP70.7, HSP66.0, HSP54.1, HSP48.6, HSP43, sHSP38.3, sHSP35.3, sHSP29, sHSP22.5, sHSP17.8 and sHSP9.5 in GS at 6h, while HKP58.7, HKP55.5 and HKP43.7 were stably overexpressed in CM, KF and PO. Temperature switching from 35°C to 8°C upregulated HKP43.4, HKP54.6, CIP14.1 and HKP19.9 for all the cultivars. The recovery process 24h subsequent to this archetype switching was governed by overexpression of small(s)HSPs of about 25.4KD-14.1KD, HKP58.7 and HKP43.5 for all cultivars. Results suggest crosstalk protection for this paradigm-shift in temperature is chiefly conferred by isoforms of constitutively expressed HKPs, CIP19.9 and CIP14.1 in S. tuberosum L. Explicitly, this differential proteome change within 22h signify HKPs may not participate in thermotolerance as HSPs, but participate in cold acclimation as CIPs, recovery as sHSPs and even switch-off during heat-stress in some cultivars as depicted in GS.