Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

The overall goal of this procedure is to isolate and extract protein from embryonic cardiac valves. This is accomplished by first isolating an embryo from a pregnant mouse. In the second step, the heart is dissected from the embryo, and then the cardiac valves are collected in the final step.

The tissue is lysed and the resulting protein sample is prepared for subsequent analysis. Ultimately, western BLO analysis can be used to assess protein expression levels. Visual demonstration of this method is critical as the dissection steps are difficult to learn because of the small compact shape of the developing heart.

At the desired embryonic day of development, use 70%ethanol to sterilize the abdomen of the pregnant female. Then lift the skin and muscle of the lower abdomen up and away from the internal organs, and dissect open the lower abdominal cavity to allow access to the uterine horn. Next, hold the cervix with forceps and carefully cut coddle to the tips.

Then lift the uterine horn cutting any connective tissue that keeps the horn in place, and complete the removal by cutting the junction of the uterine horn with the uct. Place the dissected uterine horn in a Petri dish containing cold 0.1 molar tris buffer and rinse as needed. Then cut the uterine horn.

Open lengthwise to expose the embryonic sacks. To expose an embryo, cut open an embryonic sack at the junction of the placenta and the embryo, and cut the umbilical vessels to free the embryo. Place the dissected embryo in a second Petri dish with cold 0.1 molar tris buffer, and return the first Petri dish with the remaining und dissected embryos to ice until the next embryonic dissection.

After decapitation, place the embryo on its back and cut the chest wall vertically along the side of the rib cage near a for limp and horizontally above the diaphragm. To visualize the heart, open the chest wall. Then hold the forceps high up along the great vessels, lift the heart and cut the vessels below.

Next, cut above the forceps to free the heart. If the pulmonary vessels remain uncut, remove the lungs with the heart before continuing at the embryonic stages described herein, the pulmonary artery is slightly opaque. After identifying the artery, cut and remove it above the level of the valve.

Now, cut just below the pulmonary valve, avoiding the ECD ventricular myocardium, and remove excess tris buffer away from the region of interest. Prior to placing the valve in an einor tube containing two microliters of lysis buffer like the pulmonary artery, the aorta will appear to be slightly opaque. After its identification, cut just below the aortic valve.

Again, avoiding the trabeculated ventricular myocardium. Remove any excess tri solution, and then transfer the valve to the einor tube containing lysis, buffer, and pulmonary artery tissue. After all of the tissue has been collected, immediately place the samples at minus 80 degrees Celsius until further.

Use for the protein extraction, thaw the collected tissues on ice, and then pellet the samples for one minute at 13, 100 Gs at four degrees Celsius. Check the volume of the resulting sample and use lysis buffer to bring the total volume up to 40 microliters. Then add five millimeter stainless steel beads to disrupt and homogenize the samples in a lyr for two to four minutes at 50 hertz, as per the manufacturer's instructions.

After homogenization, spin tubes briefly and transfer the samples to new tubes, leaving behind the insoluble debris for further experimental analysis. Using this preparation technique, phosphorylated smad 1 5 8 can be detected in single aortic valve regions from E 13.5 embryos. For example, the protein isolated from even a single valve region is sufficient to detect a faint p smad band with the signal intensity increasing proportionately to the number of valve regions that are pooled.

Importantly, the p smad beta actin ratio remains clearly constant across the different sample sizes due to the low levels of protein available, the same blot cannot be stripped and re probed for total smad. However, a separate blot showing total smad one and beta actin exhibits the same expression pattern. These valve regions are almost entirely devoid of contaminating ventricular myocardium using this preparation technique in combination with samples from the right ventricle, the ventricular myocardium marker, a NP is not detectable in aortic valve regions, whereas this marker is readily detected in the ventricle.

Further, the ventricular myocardial marker myosin light chain two V is also readily detectable in the ventricular sample, but is barely detectable in the aortic valve regions underscoring the preciseness of the dissection Following this dissection. Other mes like QPCR or RN AIC can be performed in order to answer additional questions like what downstream genes are affected during the different developmental steps.