MORC3 { m icr orc hidia (MORC) family CW-type zinc finger 3 , KIAA0136, ZCWCC3 and NXP-2}, is a member of the MORC protein family characterized by the conser ved domains consisting of a GHL (Gyrase B, Hsp90 and MutL)-ATPase domain at the N-terminus, a zinc finger type CW domain containing conserved four cysteines and two tryptophans, a nuclear localization signal (NLS) and coiled-coil domains at the C-terminus. There are four MORC family proteins (MORC1, MORC2, MORC3, and MORC4) in human and five (Morc1, Morc2a, Morc2b, Morc3, and Morc4) in mice. MORC1 is expressed specifically in male germ cells, whereas MORC2 and MORC3 in human are ubiquitously expressed. The autosomal recessive mutation of Morc1 in mice causes the arrest of spermatogenesis early in prophase I of meiosis. Takahashi et al . reported that MORC3 is involved in p53 activation and localization of conditionally localized p53 and constitutively localized Sp100 to promyelocytic leukemia (PML)-nuclear bodies (NBs) through its GHL-ATPase activity. MORC3 is localized in PML-nuclear bodies and entire nucleoplasm except for nucleoli.

SDS-PAGE & Western Blotting
1) Wash the 1x10e7 cells 3 times with PBS and suspend with 1 mL of Laemmli’s sample buffer.
2) Boil the samples for 2 minutes and centrifuge. Load 20 µ L of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis.
3) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for precise transfer procedure.
4) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 o C.
5) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1% skimmed milk as suggest in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on condition.)
6) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 minutes x 3 times).
7) Incubate the membrane with the 1:10,000 HRP-conjugated anti-mouse IgG diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature.
8) Wash the membrane with PBS-T (5 minutes x 3 times).
9) Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescence reagent for 1 minute. Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap.
10) Expose to an X-ray film in a dark room for 3 minutes. Develop the film as usual. The condition for exposure and development may vary.
(Positive controls for Western blotting; HL60, U937, A431, WR19L, NIH/3T3, MEF, Rat1, PC12)