Our research is focused on better understanding mitochondrial biogenesis, in particular the OXPHOS system organization and regulation. And also on defining the role that mitochondrial play in diseases from cancer to neurodegenerative disorders. Mitochondria perform multiple and essential roles in the cell, conditioning its whole metabolism and survival.
We are slowly increasing our knowledge of mitochondrial complexity and its contribution to cell adaptation mechanisms in health and disease. We have contributed to establish a new organization model for the OXPHOS system, the plasticity model. That integrates previous proposals and assumes the coexistence of super complexes and free complexes in different proportions to optimize its function and allow cellular adaptations.
Our protocol allows, starting from small tissues or cell culture samples, to obtain enough mitochondrial protein amounts to analyze super complexes assembly, status and function by blue native electrophoresis or other techniques. We can do this reproducibly in a short time with simple equipment and relatively with low costs. To begin, re-suspend the packed cell palate in a volume of hypotonic buffer equal to seven times the cell pellet volume.
Transfer the cell suspension into a Potter Dounce homogenizer and incubate on ice for two minutes, allowing the cells to swell. Break the cell membranes by performing eight to ten strokes in the homogenizer, coupled to a motor driven Teflon pestle rotating at 600 RPM. Add an equal volume of hypertonic buffer to the cell suspension to generate an isotonic environment.
Transfer the homogenate into a 10 to 15 milliliter tube. Centrifuge at 1, 000 G for five minutes at four degrees Celsius and collect the supernatant into a 1.5 milliliter polypropylene tube. To collect the mitochondrial crude fraction, centrifuge in a microfuge at 16, 000 G for two minutes at four degrees Celsius.
Discard the supernatant and re-suspend the mitochondria-enriched pellet with 0.5 milliliters of buffer A.Combine the contents of two tubes into one and centrifuge as demonstrated previously. After the last centrifugation, re-suspend the pellet in 300 microliters of buffer A.Quantify mitochondrial protein concentration using the Bradford assay. Using scissors cut the 20 to 30 milligrams of animal tissue.
Wash the tissue three to four times in homogenization buffer with the help of a strainer, taking care to avoid losing the smaller pieces. Transfer the tissue pieces with buffer to the homogenizer. For liver, spleen, and kidney tissues, perform four to six up and down strokes in the LVEM Potter with a motor-driven Teflon pestle at 600 RPM.
Begin by re-suspending the mitochondrial fractions obtained from mammalian cell cultures or tissues in blue native sample buffer to obtain a protein concentration of around 10 milligrams per milliliter. To solubilize mitochondrial membranes, add digitonin to obtain a ratio of four grams of digitonin per gram of mitochondrial protein. Mix by gentle pipetting and incubate on ice for five minutes.
Centrifuge the suspension in a microfuge at 20, 000 G for 25 minutes at four degrees Celsius to remove insoluble material. Collect the supernatant in a fresh tube, then add 5%Coomassie blue G-250, equivalent to one third of the initial re-suspension volume, and mix by pipetting. Add cold cathode A buffer into the upper chamber of the electrophoresis apparatus and anode buffer into the lower chamber.
Load 30 to 100 micrograms of mitochondrial protein into the wells on a polyacrylamide gel. After electrophoresis, place the gel in a plastic box. Add enough volume of appropriate in-gel activity assay solution to cover the gel and incubate at room temperature with gentle shaking, protecting from light.
When the appropriate bands have developed, remove the assay solution. Wash the gel twice with distilled water before fixing it with 40%methanol and 10%acetic acid for 30 minutes. Gel activity assay analysis revealed distinct super complexes assembly patterns in mice and human cells.
Free complex one was observed in mouse cells, whereas it was not detectable in human mitochondria. Complex four patterns were similar in human cell lines, but varied in mouse cell lines due to a mutant variant of SCAF1.
