Cell adhesion to proteins separated by lithium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane: a new cell-blotting technique.
Cell blotting, although conceptually simple, has failed to achieve wide practical application. Described here is a new cell-blotting technique which involves cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane at 4 degrees C. Cell bands adherent on PVDF are detected using hematoxylin, or propidium iodide (PI) staining followed by viewing under ultraviolet (UV) light. The technique allows quick microscopic visualization of adherent cells composing the bands, without requiring clearing of the membrane. Representative cell adhesion proteins from different sources, i.e., plant lectins (e.g., phytohemagglutinin, PHA; concanavalin A, ConA; and wheat germ agglutinin, WGA); extracellular matrix (ECM) proteins; and integral membrane proteins (e.g., recombinant soluble vascular cell adhesion molecule-1, rs VCAM-1) were tested for cell binding by the new cell-blotting technique using human lymphoid progenitor (NALM-6) and myeloid progenitor (KG1a) cell lines. Cell adhesion proteins retained their adhesion function in all cases tested. Specificity of cell binding on PVDF blot was demonstrated by inhibition of cell adhesion to WGA protein bands using an appropriate sugar, i.e., N-acetyl D-glucosamine. The cell blotting assay was comparable in sensitivity to Coomassie blue staining of protein bands. The ability to conduct protein extraction, separation and blotting at low temperature avoids thermal denaturation, thereby preserving the adhesion properties of the proteins. The electrophoretic/blotting system has unique detergent removal/protein renaturation properties and the ability to preserve functionally active adhesion protein complexes. The cell-blotting technique described is sufficiently robust for routine application in the investigation of novel cell adhesion proteins.
Seshi
B
B
Department of Pathology, University of Rochester School of Medicine and Dentistry, NY 14642.
D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008567:Membranes, Artificial; D011145:Polyvinyls; D011485:Protein Binding; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured; D005465:Fluorocarbon Polymers
D015815:Cell Adhesion Molecules; D008567:Membranes, Artificial; D011145:Polyvinyls; D012967:Sodium Dodecyl Sulfate; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C028913:dodecyl sulfate