A novel method of loading samples onto mini-gels for SDS-PAGE: increased sensitivity and Western blots using sub-microgram quantities of protein.
Commercially available mini-gels for sodium dodecyl sulphate (SDS)-PAGE and Western blotting are limited both by the number of lanes that can be loaded per gel and the minimum amount of protein per lane that must be loaded. Here we describe a method for loading protein samples onto existing commercially available mini-gels that allows loading of 50 or more lanes per gel. The enhanced sensitivity of the method allows Western blotting with sub-microgram quantities of protein. Samples are loaded onto filter paper strips mounted on a plastic backing sheet, and film-wrapped strips on a separate dummy loader interdigitate with the sample strips, creating a physical barrier to lateral diffusion. The sample loader sandwich is placed on top of the stacking gel, and is compatible with all commercially available SDS-PAGE systems. Comparison of 15-lane mini-gels with 30-lane micro-loader strips reveals up to a 10-fold increase in sensitivity with the new method. Using 50- and 66-lane micro-loaders, sub-microgram quantities of protein produce reliable and quantifiable signal by Western blotting. Manipulation of the ionic conditions within dummy loader strips provides a mechanism for enhancing lateral resolution, allowing for the possibility of further miniaturization.
Swank
Michael W
MW
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, United States.
Kumar
Vikas
V
Zhao
Jing
J
Wu
Gang-Yi
GY
D000906:Antibodies / Q000032:analysis; D015153:Blotting, Western / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007202:Indicators and Reagents; D008970:Molecular Weight; D036103:Nanotechnology; D009419:Nerve Tissue Proteins / Q000032:analysis*
D000906:Antibodies; D007202:Indicators and Reagents; D009419:Nerve Tissue Proteins