The use of ethanol for preserving proteins and glycoproteins of the trabecular meshwork.
We evaluated methods for storage of the trabecular meshwork and irisciliary body for protein and glycoprotein analysis. The trabecular meshwork and irisciliary body of rabbit eyes were microdissected and stored in Laemmli sample buffer, Carnoy's fluid (75% ethanol-25% glacial acetic acid), or 100% ethanol at ambient temperature, 4 degrees C, -20 degrees C, or -80 degrees C for 24 h or 30 days. Fresh and stored tissues were processed for one-dimensional polyacrylamide gel electrophoresis (PAGE) and Western blot using Con A lectin. The protein patterns of stored and fresh tissues as determined by silver-stained polyacrylamide gels were similar. However, ethanol-stored tissues revealed other proteins (MW of 15-30 kD and 150 kD), and the staining intensity and band resolution of lower MW (15-40 kD) were enhanced. The glycosylation patterns of stored and fresh tissues as determined by Con A (recognizes certain N-linked glycoproteins containing mannose and glucose) were also similar, but the ethanol-stored tissues stained more intensely, especially the high (>200 kD) and low (<35 kD) MW ranges. These PAGE results indicate that ethanol storage is useful for preserving and resolving the protein/glycoprotein profiles of the trabecular meshwork.
Higbee
R G
RG
Laboratory for Oculo-Cerebrospinal Investigation, The Children's Memorial Medical Center and Northwestern University Medical School, Department of Ophthalmology, Chicago, Illinois, U.S.A.
Knepper
P A
PA