In situ blotting: a novel method for direct transfer of native proteins from sectioned tissue to blotting membrane: procedure and some applications.
We describe a novel technique for direct transfer of native proteins from unfixed frozen tissues sections to an immobilizing matrix, e.g., nitrocellulose, polyvinyliden difluoride, or positively charged nylon membranes. Proteins are directly blotted onto the membrane, providing optimal accessibility for molecular detection but retaining the anatomic localization at the cellular level. Within 10 min a maximum protein transfer is achieved independent of the protein molecular weight. The total protein bound was 80% of the maximal binding capacity of the blotting membrane and independent of the section thickness. These results indicate that the proteins that bind to the membrane originate from the cut cell monolayer that has direct contact with the blotting membrane. This in situ blotting method provides direct protein mapping from a single cell layer of a tissue section. The procedure includes cryosectioning at 20 microns and collecting sections on a dry blotting membrane at -20 degrees C. For protein transfer the blotted sections are thawed and incubated for 10 min with Tris buffer. After incubation the sections are removed from the membrane by high-pressure spray. The blotted membranes can be subjected to several detection assays. In the present study the presence of several proteins was demonstrated in brain and thymus by immunochemical and enzyme histochemical procedures.
Okabe
M
M
Department of Animal Physiology, University of Groningen, Haren, The Netherlands.
Nyakas
C
C
Buwalda
B
B
Luiten
P G
PG
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D004798:Enzymes / Q000032:analysis*; D008297:Male; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D005465:Fluorocarbon Polymers
D004798:Enzymes; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers