Western blotting by thin-film direct coating.
A novel thin-film direct coating (TDC) technique was developed to markedly reduce the amount of antibody required for Western blotting (WB). Automatic application of the technique for a few seconds easily and homogeneously coats the specific primary antibody on the polyvinylidene fluoride (PVDF) membrane. While conventional WB requires 0.4 μg of the primary antibody, the proposed technique only uses 4 × 10(-2) μg, which can be reduced further to 4 × 10(-5) μg by reducing the coater width. Moreover, the proposed process reduces antibody probing times from 60 to 10 min. The quantification capability of TDC WB showed high linearity within a 4-log2 dynamic range for detecting target antigen glutathione-S-transferase. Furthermore, TDC WB can specifically detect the extrinsic glutathione-S-transferase added in the Escherichia coli or 293T cell lysate with better staining sensitivity than conventional WB. TDC WB can also clearly probe the intrinsic β-actin, α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase, which are usually used as control proteins in biological experiments. This novel technique has been shown to not only have valuable potential for increasing WB efficiency but also for providing significant material savings for future biomedical applications.
Institute of Applied Mechanics and ‡Department of Biochemical Science & Technology, National Taiwan University , Taipei 106, Taiwan.
Yi-Kuang
YK
Yen
Yi-Wei
YW
Jiang
Shih-Chung
SC
Chang
An-Bang
AB
Wang
D000906:Antibodies / Q000737:chemistry; D015153:Blotting, Western / Q000295:instrumentation* / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D005982:Glutathione Transferase / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry; D005465:Fluorocarbon Polymers
D000906:Antibodies; D005982:Glutathione Transferase; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers