Using two types of conventional methods to confirm protein-lipid 
interactions detected by the proteome microarrays (49).  
A. PI(4,5)P2 liposomes were first adhered to a nitrocellulose membrane, 
which was blocked by BSA; a dilution series of Rim15p, Eno2p, and Hxk1p, 
and a GST control were used to probe the membrane.  The bound proteins 
were detected using the anti-GST antibodies and an ECL kit.  
B. A reverse assay was carried out to test potential protein-lipid 
interactions.  The proteins were prepared and spotted onto nitrocellulose 
filters in a dilution series and probed with the six differentliposomes.  
As a control, the six liposomes were also added to BSA blocked membrane.  
After extensive washing, the bound liposomes were detected using an 
HRP-conjugated streptavidin and an ECL kit.  C. Linear correlation 
between the binding signals and the amounts of Rim15p in a membrane assay.  
When its liposome-binding signals from the membrane assay (Fig. 4B) were 
plotted against the concentration gradient of the spotted Rim15p, a linear 
correlation between the binding signals and the amounts of Rim15p was 
revealed.  PI(4)P showed the highest affinity to Rim15p, at least three 
times higher than the control PC.  As indicated by the chart, PI(4)P, PI(3,4)P2, 
PI(3)P, and PI(4,5)P2 binding showed a linear correlation with the Rim15p 
concentration gradient.