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Quantitative measurement of proteins by western blotting with Cy5-coupled secondary antibodies.
Fradelizi, J.; Friederich, Evelyne; Beckerle, M. C. et al.
1999In BioTechniques, 26 (3), p. 484-6, 488, 490
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Keywords :
Amino Acid Sequence; Animals; Antibodies/chemistry/immunology; Blotting, Western/methods; Calibration; Carbocyanines/chemistry; Cell Extracts/chemistry; Cell Line; Cytoskeletal Proteins; Glycoproteins; Humans; Metalloproteins/analysis/immunology; Molecular Sequence Data; Proteins/analysis; Zyxin
Abstract :
[en] The concentration of proteins in cells is an important parameter that determines how a protein will interact with other proteins or pharmacological agents. Recent developments in Western blotting techniques have now made this a method of choice to measure protein concentration in complex solutions such as total cell extracts. We show that detection of Cy5-coupled secondary antibodies by PhosphorImager analysis produces signals that approach linearity with respect to protein concentration over a 20-fold range. We used this technique to estimate cellular levels of zyxin, which is an important protein component of the actin cytoskeleton in mammalian cells. By producing specific protein standards based on sequences that are available from public databases, it is now possible to estimate the concentration of almost any protein by this technique.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Fradelizi, J.
Friederich, Evelyne ;  University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit
Beckerle, M. C.
Golsteyn, R. M.
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Quantitative measurement of proteins by western blotting with Cy5-coupled secondary antibodies.
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484-6, 488, 490 passim
Peer reviewed :
Peer reviewed
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since 18 September 2013


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