The overall goal of this biochemical procedure is to isolate intermediate filament enriched fractions from multiple mouse tissues in order to study post translational modifications of the different tissue specific intermediate filament proteins. This method can help to answer key questions in the intermediate filament field such as how the function and regulation of these important stress protective cytoskeletal proteins is affected during mammalian aging. The main advantage of this technique is that incorporating an automated tissue lysis step allows users to process multiple tissue samples in a rapid and efficient manner.
To begin, add one milliliter of ice cold Triton X-100 buffer supplemented with protease inhibitors into a glass tube homogenizer and place it on ice. Remove a small piece of tissue from liquid nitrogen storage and place it directly into the glass homogenizer. Then while keeping the homogenizer and sample on ice at all times, use approximately 50 strokes of a polytetrafluoroethylene pastille to homogenize the sample while minimizing the formation of bubbles.
Transfer the lysate to a 1.5 milliliter microcentrifuge tube on ice, and centrifuge the sample at 20, 000 times G in four degrees Celsius for 10 minutes. Next, collect the Triton X soluble supernatant fraction into a separate tube. This fraction can be used for immuno precipitation and analysis of the detergent soluble pool of IF proteins.
Then, to the tissue pellet add one milliliter of high salt buffer supplemented with protease inhibitors. Transfer it to a clean homogenizer, and dounce 100 strokes. Transfer the homogenate back to the microcentrifuge tube and place the tube on a rotating shaker in the cold room for one hour.
Centrifuge the homogenates at 20, 000 times G in four degrees Celsius for 20 minutes and discard the supernatant. Add one milliliter of ice cold PBS EDTA buffer to the pellet and transfer it to a clean homogenizer as a final cleanup step. After homogenization, transfer the sample to a new tube and centrifuge it at 20, 000 times G in four degrees Celsius for 10 minutes to obtain the IF protein rich high salt extract, or HSC.
Discard the supernatant and dissolve the pellet in 300 microliters of preheated non-reducing SDS sample buffer. Break up the pellet initially by pipetting and vortexing. And then heat the samples at 95 degrees Celsius for five minutes.
Vortex and pipette as needed, to ensure the pellet is fully dissolved which may take several minutes. Store all samples at negative 20 degrees Celsius until analysis. To carry out RNA extraction, add lysis buffer in a tube containing lysine matrix D, then add the sample tissue to the tissue lyser and pulse twice for 25 seconds each.
Separate the lysate from the matrix by centrifugation at 20, 000 times G.For protein extraction, add Triton X-100 or SDS sample buffer if preparing total lysate. Into a lysine tube with lysine matrix SS.Then, add the sample tissue. Pulse the sample briefly to mix.
To prepare HSC, use a magnet to remove the stainless steel bead from the tube, and centrifuge the tubes at 20, 000 times G in four degrees Celsius for 10 minutes. Separate the supernatant and pellet fractions and proceed as demonstrated earlier in the video. Prepare PBST buffer by adding 10 microliters of T20 to 50 milliliters of PBS pH 7.4.
Then prepare PTM antibody solution by adding one to 10 micrograms of antibody to 200 microliters of PBST. Aliquot 50 microliters of magnetic beads into a micro centrifuge tube. Place it on the magnet, and aspirate the bead storage solution.
Conjugate the beads to the immunoprecipitation antibody by resuspending the beads in the antibody solution and incubating the sample on a rotator at room temperature for 20 minutes. Place the tubes on the magnet, and aspirate the antibody solution. Then use 200 microliters of PBST to rinse the antibody conjugated beads once, and remove the solution.
Add 0.6 to one milliliter of the tissue lysate to the beads. Mix by gently pipetting, and incubate the solution on a rotator in a cold room for three hours. Place the tubes on the magnet and remove the lysate, then use 200 microliters of PBST to wash the beads five times.
After the last washing step, collect the beads and 100 microliters of PBS and transfer the beads to a new, clean tube. Place the tube on the magnet, then aspirate the PBS. Remove the tube from the magnet and add 100 microliters of non-reducing sample buffer.
Aliquot 50 microliters from the tube, and combine it with 5%2ME to make reducing samples. Heat the samples to 95 degrees Celsius for five minutes. With the tube on the magnet, collect the IP fraction from the beads, and transfer it into a fresh tube.
Store the samples at negative 20 degrees Celsius until analysis. To avoid contamination of protein samples for mass spectrometry analysis, use clean gloves to handle all gels and incubate them in clean containers that have been only washed with DD H2O. Run 20 to 50 microliters of the HSE sample on an SDS page gel according to standard conditions.
After staining the gel according to the text protocol, place the gel between plastic sheet protectors. Then scan and mark the bands that will be excised. Use a new, clean razor to excise the IF protein bands and place the bands in clean microcentrifuge tubes on ice before submitting them for mass spectrometry analysis.
This figure shows a typical result of HSEs from nine mouse tissues isolated using the rapid method. Note that this method does not work well on the pancreas and spleen, and yields additional bands in a large intestine sample. This gene expression analysis shows the Keratin 8 is up regulated during aging.
In addition, western blots show that Keratin 8 is strongly up regulated in the livers of old mice. Here, a liver HSE obtained using the automated protocol demonstrates the strong enrichment of Keratins 8 and 18 on the gel. Mass spectrometry analysis shows that K8 and K18 in the liver from an old mouse have multiple phosphorylation and acetylation sites that are not present in the liver from the young mouse.
Similar to keratin changes in the liver, analysis of brain tissue by QPCR reveals a five fold induction of GFAP mRNA in the brains of 24 month old compared to three month old mice. Finally, protein analysis of the high salt extracts by Coomassie stain, and immuno blots of total and acetyl lysine enriched fractions reveals that GFAP protein is up regulated and acetylated in the aged mouse brain. Following this procedure, other methods such as mass spectrometry based proteo mix can be used to answer additional questions such as what are the important post translational modifications and binding partners of intermediate filament proteins during different physiological and pathophysiological conditions.
After watching this video, you should have a good understanding of how to isolate intermediate filament proteins from different mammalian tissues.
