A thin‐layer isoelectric focusing method for the separation of proteins in follicular fluid and seminal plasma

Abstract

AbstractA thin‐layer electrofocusing method has been developed for the separation of proteins in human follicular fluid, seminal plasma, and blood plasma/serum. The method has a number of unique features. It is simple and rapid, and it produces distinct, undistorted bands that can be readily quantitated. Any samples solubilized by standard methods can be analyzed, and it can be adapted to favor specific regions of the pH spectrum. The gels used are composed of agarose and sorbitol, and they are formed between a plastic template and a Mylar film. Sample volumes ranging from 0.5 to 5 μl can be used. Protein separation is usually completed in 30–40 min at constant power of 1 or 1.5 W. The use of low power ensures that little heat is generated during the run and hence only minimal temperature control is needed. Carbon rod electrodes are a special feature as they allow the inverted gel to rest in direct contact with the electrodes, eliminating the necessity for electrode solutions. The inverted gel position allows fluid to drain from the surface of the gel and so prevents distortion of protein patterns. This overcomes the problems inherent in conventional horizontal electrofocusing of unpurified biologic fluids such as plasma, follicular fluid, and seminal plasma. Rapid protein staining is carried out with fixed and dried gels. The excellent resolution and the lack of distortion make this method suitable for reliable quantitative analysis of differences between samples by means of laser beam densitometry. This has enabled us to assess differences in specific follicular fluid proteins between individual follicles in the human, and to correlate these differences with fertilizability and cleavage potential of oocytes derived from such follicles.