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

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.