Protein Protocols Protein Protocols

Staining with MDPF 375
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MDPF Staining of Proteins on Western Blots
F. Javier Alba and Joan-Ramon Daban
1. Introduction
We describe a method for the detection of total protein patterns on polyvinylidene
difluoride (PVDF) membranes using the fluorogenic dye 2-methoxy-2,4-diphenyl-
3(2H)-furanone (MDPF) (1). This method is based on the fluorescent properties of this
dye (2). As can be seen in Fig. 1, MDPF (A) and the hydrolysis product (C) are nonfluorescent;
only the adduct B formed with the proteins is fluorescent. This makes
unnecessary the destaining of the PVDF membrane after protein labeling. The whole
process of staining with MDPF is completed in about 20 min. Wet membranes are
translucent, allowing the visualization of MDPF labeled protein bands by transillumination
with UV light (see Fig. 2A). Electrophoretic bands containing less than 10 ng of
protein transferred to PVDF membranes can be detected after the reaction with MDPF.
This staining method is compatible with previous visualization of protein bands on
the sodium dodecyl sulfate (SDS)-polyacrylamide gel with the noncovalent fluorescent
dye Nile red (see Chapter 30). Thus, Nile red and MDPF staining can be performed
sequentially. This allows the rapid monitoring of total protein patterns on both
the electrophoretic gel and Western blot. In addition, MDPF staining allows further
immunodetection of specific bands with polyclonal antibodies (see Fig. 2B). Finally,
using the adequate conditions described in the Materials section, MDPF staining does
not preclude the N-terminal sequence analysis of proteins in selected bands.
2. Materials
All solutions should be prepared using electrophoresis-grade reagents and deionized
water and stored at room temperature. Wear gloves to handle all reagents and solutions
and do not pipet by mouth. Collect and dispose all waste according to good laboratory
practice and waste disposal regulations.
1. MDPF: Concentrated stock (35 mM) in dimethyl sulfoxide (DMSO). This solution is stable
for 1 wk at room temperature in a glass bottle wrapped in aluminum foil. Handle this
solution with care, DMSO is flammable and, in addition, this solvent may facilitate
the passage of hazardous chemicals such MDPF through the skin. MDPF can be
obtained from Fluka (Bunch, Switzerland).
2. PVDF membranes (Bio-Rad Laboratories [Hercules, CA]).
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
375