Two-Dimension Affinity Resolution Electrophoresis Demonstrates that Three Distinct Heparin Populations Interact with Antithrombin III

Authors
Edens, R.E.
Fromm, J.R.
Fromm, S.J.
Linhardt, Robert J.
Weiler, J.M.
ORCID
https://orcid.org/0000-0003-2219-5833
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Other Contributors
Issue Date
1995
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
Degree
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Full Citation
Two-Dimension Affinity Resolution Electrophoresis Demonstrates that Three Distinct Heparin Populations Interact with Antithrombin III R. E. Edens, J. R. Fromm, S. J. Fromm, R.J. Linhardt, J. M. Weiler, Biochemistry, 34, 2400-2407, 1995.
Abstract
Tween 20 is frequently used in blotting analysis of proteins and nucleic acids to decrease background staining due to nonspecific binding of antibodies to various membranes. Inclusion of 0.1% (w/v) Tween 20 in the washing buffer (Buffer 1) significantly decreased the chemiluminescence signals of both LMW-EGF and HMW-EGFs (data not shown). This result indicates that the binding of proteins to PVDF membranes is susceptible to the detergent even after fixation with formaldehyde. In conclusion, we suggest the following conditions for the analysis ofLMW-EGF: (a) treat PVDF membranes with a 500 J.tg/ml gelatin solution, (b) carry out electroblotting for 10 min (the duration depends on the blotting conditions used), (c) fix the membranes with formaldehyde after blotting, and (d) do not include Tween 20 in the washing buffer. The optimized conditions made it possible to detect 0.1-0.3 ng of rat LMWEGF (Fig. 3). AJthough the conditions are not optimum for the detection of HMW-EGFs, as described above, it is possible to analyze LMW- and HMW-EGF species simultaneously on a single membrane with this method.
Description
Biochemistry, 34, 2400-2407
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Department
The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
Publisher
Relationships
The Linhardt Research Labs Online Collection
Rensselaer Polytechnic Institute, Troy, NY
https://harc.rpi.edu/
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