Identification of human hsp-70 expression in stably transfected rodent cells by isoelectrofocus immunoblots Journal Article


Authors: Liu, R. Y.; Li, G. C.
Article Title: Identification of human hsp-70 expression in stably transfected rodent cells by isoelectrofocus immunoblots
Abstract: The major heat-shock protein, hsp-70, is synthesized by cells from a wide variety of organisms in response to heat shock or other stresses. It is assumed that hsp-70 may have an important thermal protective function. To test this hypothesis directly, we have transfected rat fibroblast cells with appropriate expression plasmids containing a cloned human hsp-70 gene. Stable transfectants expressing the human hsp-70 gene product were identified by Western blot analysis. During the course of selecting successful transfectants, we found that when standard methods of sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and immunostaining were employed with the monoclonal antibody raised against the human hsp-70 antigen, we were unable to differentiate the human hsp-70 from the heat- inducible rat hsp-70. This was because the monoclonal antibody cross-reacts with the human and rat proteins, which have the same mobility in SDS-PAGE, and it is difficult to determine which protein is expressed. To improve the resolution of the Western blot technique, we performed additional immunoblot analysis of cellular proteins separated by slab gel isoelectrofocusing. Our study shows that the isoelectrofocusing technique, when combined with antibodies against hsp-70, gave a better resolution for the separation of exogenous human hsp-70 and the endogenous rat hsp-70 than the commonly used SDS-PAGE Western blot analysis. It provides a rapid and specific method to identify positive colonies that express the human hsp-70 gene in transfected rodent cells.
Keywords: controlled study; human cell; nonhuman; protein analysis; animal cell; animal; gene expression; cell protein; gene product; transfection; animalia; monoclonal antibody; genetic transfection; rat; immunoblotting; cell line, transformed; rats; rodentia; sequence homology; cross reaction; polyacrylamide gel electrophoresis; heat-shock proteins; heat shock protein; isoelectric focusing; dodecyl sulfate sodium; human; priority journal; article; heat stress; support, u.s. gov't, p.h.s.
Journal Title: Radiation Research
Volume: 129
Issue: 3
ISSN: 0033-7587
Publisher: Radiation Research Society  
Date Published: 1992-03-01
Start Page: 357
End Page: 361
Language: English
DOI: 10.2307/3578037
PUBMED: 1542724
PROVIDER: scopus
DOI/URL:
Notes: Source: Scopus
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  1. Gloria C Li
    132 Li