Improved Alignment and Quantification of Protein Signals in Two-Dimensional Western Blotting

Western blotting is widely used for
protein identification and
quantification in research applications, but different protein species,
resulting from alternative splicing and post-translational modifications,
can often only be detected individually by two-dimensional gel electrophoresis
and immunodetection by Western blotting (2D-WB). The additional separation
by isoelectric focusing enables the detection of different protein
species with the same specific antibody. Reliable assignment of signals
from antibody-based detection to the total protein spot pattern of
the original gel image is a challenge in 2D-WB, often resulting in
ambiguous results. We therefore propose a reliable strategy for assignment
of antibody signals from 2D-WB to the total protein spot pattern,
using an imaging workflow in combination with a straightforward and
easily reproducible image alignment strategy. The strategy employs
vector-based alignment of protein spots and image contours in a stepwise
manner. Our workflow is compatible with various protein visualization
techniques, including prelabeling of proteins and poststaining of
gels and membranes, as well as with chemiluminescent and fluorescent
detection of bound antibody. Here, we provide a detailed description
of potential applications and benefits of our workflow. We use experimental
test settings with gold-standard stressors in combination with multiple
staining and detection methods, as well as spike-in recombinant proteins.
Our results demonstrate reliable attribution of signals to very similar
heat shock proteins, phosphorylation patterns, and global analysis
of proteins modified with O-linked N-acetylglucosamine (O-GlcNAc).