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[Establishment of two-dimensional gel electrophoretic protein map of human spermatozoa]
1Human Reproductive Engineering Laboratory, Hunan Medical College, Central South University, Changsha 410078, China.
Objective:
To establish the method of 2-dimensional electrophoresis(2-DE) for proteins of human spermatozoa and to construct a protein map of human spermatozoa.
Methods:
The sperm pellet was prepared with simple Percoll layer protocol. We studied the effects of various sample preparation methods, loading quantities and isoelectric-focusing protocols on the quality of silver-stained 2-DE map, and constructed a primary protein map of human spermatozoa.
Result:
Up to 703 protein spots were acquired with sample preparation Method I while only 194-210 spots with Method II. With immobilized pH gradients and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(IPG-DALT) we could acquire over 700 spots while only 280-300 with isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(ISO-DALT).
Conclusion:
It is satisfactory to lyse sperm with sample preparation Method I and to separate sperm proteins by IPG-DALT for establishing 2-D map of human sperm.