Enucleation for Western Blot Analysis

Although the aforementioned enucleation protocol is suitable for obtaining healed enucleated oocytes for functional studies, it is not recommended in studies in which the nucleus and enucleated oocyte are to be analyzed simultaneously. This is because the nucleus extruded into the half-strength OR2 will swell and become very fragile. The alternative enucleation method described in the following paragraph was originally developed by Gall et al. (12). The advantages of this protocol are as follows: (1) Gall's medium is designed to maintain the nuclear content as a gelatinous ball even after the removal of the nuclear envelope, so one can expect minimum loss of nuclear contents during the enucleation process; (2) the cytoplasm of the enucleated oocyte will remain very stiff in Gall's medium and will not be extruded, so minimum loss of cytoplasm can be achieved. It should be noted that neither the enucleated oocytes nor the nuclei isolated in this manner can be used for further functional assays.

1. Prepare two pairs of forceps with different tip conditions: One pair with bent and blunt tips is used to hold the oocyte; another pair with bent and sharp tips is used to snip a small opening on the animal pole.

Fig. 4. Enucleation and healing: (A) A single oocyte after 30-min incubation in half-strength OR2 medium. (B) The extrusion of the nucleus. (C) The extrusion of cytoplasm after ejection of the nucleus. (D) Excessive loss of cytoplasm. (E) A well-healed oocyte after 1.5-h incubation in the healing medium, with a small piece of cytoplasm still attached. (F) A poorly healed oocyte with a big hole after 1.5-h incubation in the healing medium. (G) A well-healed oocyte after 2-h incubation in OR2 medium with gentamicin and PVA. (H) An oocyte that is not completely healed after 2-h incubation in OR2 medium with gentamicin and PVA; such oocytes were discarded. L

Fig. 4. Enucleation and healing: (A) A single oocyte after 30-min incubation in half-strength OR2 medium. (B) The extrusion of the nucleus. (C) The extrusion of cytoplasm after ejection of the nucleus. (D) Excessive loss of cytoplasm. (E) A well-healed oocyte after 1.5-h incubation in the healing medium, with a small piece of cytoplasm still attached. (F) A poorly healed oocyte with a big hole after 1.5-h incubation in the healing medium. (G) A well-healed oocyte after 2-h incubation in OR2 medium with gentamicin and PVA. (H) An oocyte that is not completely healed after 2-h incubation in OR2 medium with gentamicin and PVA; such oocytes were discarded. L

2. Under a dissecting microscope, place a small group of oocytes (fewer than 10) in a Petri dish containing Gall's medium (plus Mg2+). Use the sharp forceps to snip a small opening on the animal pole. Use both forceps to squeeze the nucleus out of the oocyte (see Note 6).

3. If the nuclei contain much cytoplasm, transfer them to another Petri dish containing Gall's medium and wash away the attached cytoplasmic fragments by blowing them gently using a P2 Pipetteman. Otherwise, transfer the nuclei to an Eppendorf tube containing 0.5 mL ice-cold nucleus medium. If the enucleated oocytes are also needed, transfer them to another tube (see Note 7).

4. Immediately centrifuge the tube containing nuclei at 400g in a refrigerated Eppendorf centrifuge (4°C) for 30 s, carefully aspirate the supernatant without disturbing the nuclei pellet, and then add 2X sodium dodecyl sulfate sample buffer.

5. Aspirate excess medium from the tube containing the enucleated oocytes, lyse the enucleated oocytes in ice-cold phosphate-buffered saline lysis buffer (10 nL per oocyte; lysis by passing the oocytes through a yellow pipette tip), centrifuge at 10,000g in a refrigerated Eppendorf centrifuge (4°C) for 5 min, and recover the supernatant and mix it with an equal volume of 2X sodium dodecyl sulfate sample buffer.

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