This protocol provides a step-by-step procedure to sample the perivitelline membrane sublayers from avian eggs for further investigation of their physiological role in bird reproduction. The sampling of the perivitelline membrane has been optimized at each step to limit any structural and molecular damages. The protocol was further developed for in-depth proteomics.
Begin by sampling the perivitelline layer or PL from a freshly laid unfertilized egg. Break the egg and use an egg separator to separate the yolk from the white. Remove the chalazae with small scissors and roll the yolk over a filter paper to remove adherent albumin that appears as a transparent but visible structure.
Immerse the yolk in a crystallizer containing 10 millimolar Tris HCL at pH eight that has been previously cooled to four degrees Celsius and remove the PL area over the germinal disk within a one centimeter zone using blunt scissors. Rupture the PL with small scissors inside the buffer, then hold the two edges of the ruptured PL with forceps and peel it off the yolk. Rinse the PL several times in baths of 10 millimolar Tris HCL until no trace of yolk is visible.
Ensure that the PL is clean, white, and floating in the buffer. Process the PL for sublayer separation or proceed directly to biochemical analyses. To separate the OPL and IPL, spread the entire sample with the OPL facing up in a plastic Petri dish filled with 10 millimolar Tris HCL and 50 millimolar sodium chloride and maintain it flat with as few wrinkles as possible.
Determine the location of the remaining chalazae that are only attached to the OPL. Then cut the entire PL into pieces of about two by three centimeters with small scissors. Mechanically separate the two layers with ultra precise tip forceps under a dissecting microscope.
Store the resulting IPL and OPL samples individually in micro tubes at negative 80 degrees Celsius until further use. To perform primary protein solubilization, freeze dry the IPL and OPL samples individually to remove remaining water, then cut approximately one milligram from each sample and place it inside a clean micro tube with a leak-proof screw cap. Keep the remaining samples in tightly closed tubes for prolonged storage at negative 20 or negative 80 degrees Celsius.
Mix one milligram of each lyophilized sublayer with 400 microliters of 50 millimolar Tris at pH seven and 500 millimolar sodium chloride. Use a mixer mill twice for five minutes at 30 Hertz to disintegrate the structures into micro particles and facilitate protein solubilization. Collect 400 microliters of the samples into two clean micro tubes.
To prepare the samples for electrophoresis, add 5X SDS-PAGE sample buffer to each 400 microliter sample and heat it to 100 degrees Celsius for five minutes. Load a maximum of 20 micrograms of proteins into each lane of a 4-20%gradient SDS polyacrylamide gel and perform electrophoresis at 120 volts. After electrophoresis, remove the gel from the glass plates and stain it with Coomassie Brilliant Blue solution for 30 minutes, then de-stain with a solution consisting of 50%water, 40%ethanol and 10%acetic acid until the gel background appears light blue.
Transfer the gel into a Petri dish containing deionized water for de-staining and re-hydration. Proteins should appear as blue bands with a transparent background. The PL was subjected to various buffers to identify conditions allowing minimal protein loss and optimal sublayer separation.
Protein released at varying Tris concentrations, pH, and sodium chloride concentrations are shown here. The resulting two sublayers were observed under a dissecting microscope. IPL is translucent while OPL is dense, cloudy, and whitish.
Following separation, OPL and IPL were lyophilized independently and their protein content was completely dissolved using a combination of mechanical grinding, an anionic detergent, a reducing agent, and boiling. The samples exhibited distinct electrophoretic profiles on a polyacrylamide gel. When attempting this protocol, keep in mind that separation of the sublayers is a critical step of the procedure and must be performed using a binocular microscope in a buffer containing a minimal concentration of salt.
To further elucidate their respective physiological function, the perivitelline membrane sublayer samples may be analyzed for histological characterization using electronic microscopy and for functional studies using imaging assays and cell migration.
