The overall goal of this procedure is to isolate the mitochondrion, endoplasmic reticular membrane, micro domains, or MAMs from mouse brain purified MAMs are further extracted to obtain the glyco finger lipid enriched micro domain fractions or gems. This is accomplished by first dissecting the mouse brain, which is then homogenized in a specific buffer using a dance homogenizer. The resulting homogenate is centrifuged at low speed to remove nuclei, unli cells, and day debris.
The second step is to prepare a crude mitochondrial fraction. Through further centrifugation of the supinate, the resulting pellet is resuspended and further separated using a discontinuous sucrose gradient. Next, the crude mitochondrial fraction is applied to a peral gradient.
After centrifugation and several washing steps, the pure mitochondrial fraction and the MAMs are obtained. The final step is to treat the MAMs with a solution containing TRITTON X 100 to extract the glyco finger lipid enriched micro domain fraction gem. Ultimately, the purity of the fractions is validated by western blos analyses by using antibodies against protein markers that a characteristic of the various organelle fractions.
The main advantage of this procedure of an existing methods is that it has been adapted to the isolation of mitochondria mam and gem fractions from adult mouse brain using relatively small amount of study material. This optimized procedure is very reproducible and enables the isolation of sufficient quantities of the different organal fractions to be used by researchers, especially those in the neurobiology field. For the analysis of their proteins of interest, demonstrating the procedure will be a a, a technician from my laboratory.
After removing the brain from a carbon dioxide euthanized mouse, begin this procedure by cutting the brain in half. Place each half into a separate two milliliter tube on ice. Weigh the tubes using an empty pre chilled two milliliter tube to zero the balance and store them on ice.
Transfer half of the brain into a pre chilled two milliliter glass down tissue grinder containing one milliliter of cold fractionation solution. A homogenized with 15 total strokes of a large clearance pestle. Place the homogenate in a sterile 15 milliliter Falcon tube on ice and dilute up to 10 volumes of cold fractionation solution A based on the weight of the sample.
Then centrifuge the sample at 1, 400 times G for 10 minutes of four degrees. SIUs carefully remove the supinate taking care not to disturb the pellet and transfer to a 30 milliliter round bottom glass centrifuge tube on ice. Next, resus, suspend the pellet in the same 10 volumes of fractionation solution.
A homogenize the resuspended pellet in the same grinder, one milliliter at a time. This time use three to six strokes of a small clearance pestle. Combine the homogenous in a 15 milliliter Falcon tube and centrifuge at 710 times G for 10 minutes at four degrees Celsius.
This results in appellative nuclei and cell debris. Carefully remove the sup natant and pull it with the previous supine natant saved from the earlier centrifugation to begin mitochondrial isolation centrifuge the S supernatants at 13, 800 times G.The 10 minutes of four degrees Celsius. Then transfer the snat to an UltraClear Beckman centrifuge tube on ice.
Re suspend the pellets containing the mitochondria fraction in 10 volumes of fractionation solution A and homogenize one milliliter at a time. This time use three to six strokes of a small clearance Pele, combining the homogenates in a new 30 milliliter round bottom glass centrifuge tube on ice. Further purify the sample by repeating these steps one more time.
Centrifuge the homogenate. Pull the S supernatants and re homogenize the cell pellet one milliliter at a time after suspending it in 10 volumes of solution A.The next step is to resuspend the pellet containing the enriched mitochondrial fraction in 4.8 milliliters of fractionation solution B per gram of original brain weight homogenize using six strokes of a small clearance pestle in a fresh homogenizer store this homogenate on ice. Then prepare a discontinuous sucrose gradient in an UltraClear Beckman centrifuge tube.
First, add the resuspended palette followed by three milliliters of 850 millimolar sucrose, three milliliters of one molar sucrose, and finally three milliliters of 1.2 molar sucrose. Be sure to add the solution slowly from the bottom to ensure that no bubbles are formed. Finally, centrifuge at 82, 500 times G for two hours at four degrees Celsius.
The resulting separation will produce three bands and appellate. The appellate contains the crude mitochondria that will be used in the subsequent purification steps. Reus bend the freshly isolated crude mitochondria from one brain half with two milliliters of isolation medium.
Next, add eight milliliters of a 30%per core gradient to a fresh UltraClear Beckman centrifuge tube. Layer the mitochondrial suspension on top of the prepared gradient very slowly to avoid any bubble formation. Then centrifuge at 95, 000 times G for 30 minutes at four degrees Celsius following gradient separation.
First, remove the heavy fraction, which is the lower band with a glass pasta pipette, and transfer it to a fresh round bottom glass tube. Store the tube on ice. Next, remove the light fraction, which is the upper band with a new glass pasta pipette and transfer to a separate fresh round bottom glass tube.
Place this tube on ice as well. Dilute both fractions with 10 milliliters of isolation, medium and centrifuge at 6, 300 times G for 10 minutes at four degrees Celsius. Discard the supinate from the heavy fraction and re suspend the pellet with another 10 milliliters of isolation.Medium.
Centrifuge the sample again with the same conditions. The resulting pellet will be the pure mitochondrial fraction. Next, transfer the supinate from the light fraction to an UltraClear Beckman centrifuge tube on ice.
The pellet from this fraction is discarded as it contains contaminating mitochondria. Add sufficient isolation medium to the supinate from the light fraction to fill the tube and centrifuge at 100, 000 times G for one hour at four degrees Celsius. The resulting pellet will contain the mitochondria associated ER membranes or MAMs.
Carefully remove and discard the supinate. Begin extraction of the glyco finger lipid enriched micro domains or gems by izing the MAM's fraction in 500 microliters to one milliliter of extraction buffer on ice for 20 minutes. Next, centrifuge the lysate 15, 300 times G for two minutes of four degrees Celsius.
Collect the sup natant and centrifuge the pellet for two minutes to remove all remaining tritons 100 soluble material, and finally resuspend the pellet with the solubilizing buffer. This solubilized material represents the gem fractions and can be used for further analysis. Western blots were run to check the purity and distribution of markers for endoplasmic reticulum, cytosolic proteins, and mitochondrial markers in the cytosolic fraction.
The endoplasmic reticulum fraction, the pure mitochondrial fraction and the MAMs fraction. Western blots also showed that the gems were positive for cavi in one, a marker of cli and negative for endoplasmic reticulum and mitochondrial proteins. On the other hand, the Triton X 100 extracted MAMs were positive for endoplasmic reticulum and mitochondrial markers and showed little cavi in one While attempting this procedure.
It is important to remember to use freshly dissected brain tissue and to subsequently keep all solutions samples and tubes on ice. Also, it is recommended to save a small QUT of sample from each step of the fractionation to be able to determine via immuno blotting the overall success of the purification procedure.
