This protocol can be used to gain greater insight into the dynamics and roles of phosphoproteins active during mammalian craniofacial development. This protocol overcomes the common barrier of dephosphorylation during protein isolation from two physiologically relevant context, mouse facial processes and cultured mouse embryonic palatal mesenchyme cells. It is imperative to move quickly while keeping all reagents and materials on ice and to use phosphatase inhibitors in all lysis buffers to maintain the integrity of phosphorylated proteins.
To begin, lay the mouse body on a dissecting board with the ventral side facing up, and spray the mouse abdomen with 70%ethanol. Then open the abdominal cavity by pinching and lifting the skin anterior to the vaginal opening with straight Semken forceps. Next, cut the lifted skin in underlying layers with straight blade surgical scissors at a 45 degree angle on either side to generate a V-shape that extends to each lateral surface roughly halfway between the four limbs and hind limbs.
Using the Semken forceps, grip one of the uterine horns and cut below the oviduct and above the cervix with surgical scissors. To allow for complete removal of the uterine horn, cut away the mesometrium, then transfer the dissected uterine horn to 10 milliliters of histology PBS in a 10 centimeter Petri dish. Similarly, remove the second uterine horn on the opposing side of the abdominal cavity.
Afterward, place the 10 centimeter Petri dish containing both uterine horns on ice. Under a dissecting stereo microscope, carefully dissect out each embryo from the uterine horns with Dumont 5 fine forceps. Then slowly pull away the myometrium, decidua, and chorion.
Next, tear and remove the relatively transparent amnion surrounding the embryo and sever the umbilical cord connecting the embryo to the placenta. Then transfer each dissected embryo to 2.5 milliliters of histology PBS in an individual well of a 12-well cell culture plate on ice using a cut plastic transfer pipet. Prepare three 10 centimeter Petri dishes containing 10 milliliters of histology PBS and keep them on ice to be used in rotation between embryos.
Then transfer one embryo from an individual well of the 12-well cell culture plate to one of the 10 centimeter Petri dishes with histology PBS on ice using a cut plastic transfer pipette. Under the dissecting microscope, separate each maxillary process from the face using the fine forceps by first making a cut at the anterior side of one maxillary process along the natural indentation separating the lateral nasal process in the maxillary process. Next, cut the posterior side of the maxillary process along the natural indentation separating the maxillary process and the mandibular process.
Then completely separate the maxillary process by making a vertical cut from the anterior to posterior sides of the maxillary process on the eye side of the maxillary process where the natural indentations referenced above end. Using a nine inch Pasteur pipette with a two milliliter small latex bulb, transfer the dissected pair of maxillary processes in a small droplet of approximately 30 microliters of histology PBS to a labeled 35 millimeter Petri dish on ice. Then transfer the pair of maxillary process tissues to a labeled 1.5 milliliter microcentrifuge tube on ice using the Pasteur pipette, minimizing the transfer of histology PBS.
Further, remove any excess histology PBS in the 1.5 milliliter microcentrifuge tube with the Pasteur pipette. Add 0.1 milliliter of ice cold NP40 lysis buffer with protease and phosphatase inhibitors added immediately before use on ice. Then pipette up and down 10 times with a 200 microliter PIPETMAN.
Next, vortex for 10 seconds, and then pipette up and down 10 times with a 200 microliter PIPETMAN, while avoiding the generation of bubbles. Incubate at four degrees Celsius for two hours while rotating end-over-end using a 1.5 milliliter or two milliliter paddle with a tube revolver. Then centrifuge the samples at 13, 500 x g for 20 minutes at four degrees Celsius and collect the supernatant to a new 1.5 milliliter microcentrifuge tube on ice with a 200 milliliter PIPETMAN.
First, aspirate the medium from the mouse embryonic palatal mesenchyme cells using a 5.75 inch Pasteur pipette attached to a vacuum system. Then wash the cells twice with ice cold tissue culture PBS and tilt the plate to the side during the last wash to ensure all the tissue culture PBS is aspirated. Next, add ice cold NP40 lysis buffer with protease and phosphatase inhibitors added immediately before use on ice to lyse the cells.
Incubate the plate on ice for five minutes with rotation approximately every minute to ensure complete coverage of the plate. Then scrape the cells off the plate using a pre-cooled cell lifter, and transfer the cell suspension to a pre-cooled 1.5 milliliter microcentrifuge tube on ice. Afterward, incubate the cell suspension at four degrees Celsius for 30 minutes while rotating end-over-end using a 1.5 milliliter or two milliliter paddle with a tube revolver.
Then centrifuge the samples at 13, 500 x g for 20 minutes at four degrees Celsius and collect the supernatant to a new 1.5 milliliter microcentrifuge tube on ice with a 200 milliliter PIPETMAN. Western blot analysis of whole cell lysates from immortalized mouse embryonic palatal mesenchyme cells following stimulation with PDFG-B ligand from 2 to 15 minutes revealed distinct reproducible bands for phosphoproteins that run at or near the height of the corresponding total protein band. An increase in phosphoprotein band intensities was observed upon treatment with a growth factor compared to those of untreated cells, while total protein band intensities were relatively equal between samples.
This protocol can be used to probe how perturbations and phosphorylation directly influence intercellular signaling, gene expression, and cellular activity in craniofacial contexts.
