Our new protocol helps to distinguish and quantify three statuses of tube rings, such as stable microtubules, labile microtubules, and free tubules in animal tissues. This technique is composed of simple steps and can evaluate the structural stability of tubing, which cannot be detected by quantification of tubing post translational modification. This method is essential for the research and development of therapeutic ways for diseases where microtubule stability is critical, such as Alzheimer's disease and cancers.
Start the procedure by preparing the lab wear for tissue dissection. Fill a box with crushed ice and place two Petri dishes on it. Fill one dish with ice cold phosphate buffer solution or PBS for transient wash and storage of dissected tissues.
Lay filter paper moistened with PBS on the second dish. Next, weigh the 1.5-milliliter microtubes filled with PBS for dissected tissue storage. After extracting the tissues from a euthanized mouse, transfer them to the tubes and re-weigh each microtube.
Move the tissues into a glass homogenizer with ice-cold microtubule stabilizing buffer or MSB+medium, and homogenize the tissue immediately using a chilled homogenizer. Now transfer the tissue homogenate to a two-milliliter microtube using a Pasteur pipette and centrifuge at 2, 400 G for three minutes at two degrees Celsius. Transfer the supernatant to a new 1.5-milliliter microtube to remove the debris.
Vortex the supernatant or the S1 fraction in a new 1.5-milliliter microtube. Immediately pipette 200 microliters of the S1 fraction into a centrifugation microtube and centrifuge at 100, 000 G using a TLA-55 rotor for 20 minutes at two degrees celsius. After the second centrifugation, separate the S2 supernatant from the P2 precipitate.
Immediately transfer the whole amount of the S2 fraction into a centrifugation microtube and use a TLA-120.2 rotor to spin it at 500, 000 G for 60 minutes at two degrees Celsius. After the third centrifugation, separate the S3 supernatant from the P3 precipitate. Dissolve the entire S3 fraction in 200 microliters of 2X sodium dodecyl sulfate, or SDS sample buffer.
Mix the remaining S1 fractions with an equal volume of 2X SDS sample buffer for use as a standard curve for western blotting. Next, add 400 microliters of SDS sample buffer to the P2 and P3 fraction tubes. Then, briefly sonicate the solution to dissolve the precipitate before transferring the samples into new 1.5-milliliter microtubes and placing them in the icebox.
Boil all the samples at 100 degrees Celsius for three minutes. Microtubules in the P2 fraction accounted for 34.86%of the total alpha-tubulin in a mouse brain, whereas those in the P3 and S3 fractions accounted for 56.13%and 9.01%respectively. The percentage of beta-3 tubulin in the P2, P3, and S3 fractions were not significantly different from those of alpha-tubulin.
The S2 fraction showed limited passage of tubulin through a 300-kilodalton ultrafiltration spin column, while the S3 fraction allowed for complete passage of tubulin complexes. Chromatographic separation resulted in a 100-kilodalton elution of S3 tubulin. Equal proportions of alpha-and beta-tubulin were recovered in each fraction.
P2 fractions were significantly enriched with acetylated alpha-tubulin, While tyrosinated alpha-tubulin was dominant in the P3 fraction. Freezing the brain prior to homogenization resulted in decreased alpha-tubulin concentration in the P2 fractions. However, alpha-tubulin increased in the P3 fraction.
Freezing also decreased the acetylation levels and increased tyrosination levels of alpha-tubulin in the P2 fraction. Nocodazole treatment decreased alpha-tubulin in the P2 fraction. In contrast to freezing, nocodazole did not affect the P2 tubulin post-translational modifications.
The brain tissue exhibited a significantly higher concentration of P2 tubulins, whereas P3 tubulins were concentrated in proliferative tissue. Western blotting showed that the P2 tubulin specifically found in the nervous system originated from stable microtubules, and P3 tubulin originated from labile microtubules. Microtubules stability is highly dynamic.
It is important to complete this protocol quickly, without interruptions, in the cold temperature environment. Also ensure that the tissues and fractions have not been frozen until being dissolved in SDS sample buffer. Combining immune presentation using each fraction with proteomics is expected to identify factors involved in microtubule dynamics.
Using this method, we revealed how normal tau exists on microtubules. It can also be used to study how it becomes abnormal in aged brains in order to identify new drug targets of dementia.
