The overall goal of this procedure is to prepare translationally active synap to neuro zones from mouse brain cortex, using a discontinuous peral sucrose density gradient. This is accomplished by first collecting and homogenizing the mouse cortices, and then centrifuging the homogenate. Following this, apply the centrifuge homogenate, or sup natant to the preport per co sucrose gradients.
The final step is to centrifuge and collect the synaptosome fraction. Ultimately, western blot analysis and S 35 methionine incorporation experiments show that the synaptosome fraction is synaptically enriched and translationally active. The main advantages of this technique over existing methods like centrifugation and filtration, our first mechanical damage is avoided by utilizing density gradients, and second per call has low toxicity towards cells in their constituents.
Generally, individuals new to this method will struggle with this protocol because timing plays a key role. Once the cortis have been harvested, the procedure should be rapidly completed. To begin the procedure, prepare the 3, 10, 15, and 23%gradient layers by adding the respective amounts of SIP as described in the accompanying manuscript to GM buffer and mix well.
Pour gradient layers by pipetting two milliliters from each of the 23 15, 10, and 3%isosmotic perol solutions into the Beckman centrifuge tubes with caps. Using a P 1000 pipette, the interface between the layers should be clearly discernible with no mixing of the layers. Store the gradients of four degrees Celsius for at least 20 minutes before use.
Before starting this procedure, prepare other necessary solutions as described in the accompanying manuscript. Euthanize the mouse at the age of P 13 to P 21 and prepare it for dissection by spraying the back of the neck and head with 70%ethanol. Then use a pair of sharp scissors to cut through the spinal cord at the base of the skull.
Next, remove the skin from the top of the skull. Cut the skull laterally between the parietal bone and the inter parietal bone, or between the cerebrum and the cortex regions. After that, cut the skull from the base to the nose along the sagittal suture.
At this step, carefully remove the soft parietal bone by pulling each hemisphere to the side. After that, insert the spatula into the brain above the cerebellum to scoop out the cortex. Place it in ice cold GM buffer, and repeat the procedures again for collecting more cortices.
Now rinse the cortices in ice cold GM buffer. Then transfer two cortices to a glass down homogenizer containing five milliliters of cold GM buffer. Gently homogenize the cortices with five to 10 strokes of pestle A, the loose pestle followed by five to 10 strokes of pestle B, the type pestle.
The number of strokes varies depending on the individual homogenizer. Transfer the homogenate to a 15 milliliter conical tube. Centrifuge it at 1000 times.
Gravity for 10 minutes at four degrees Celsius in an Allegra six KR centrifuge to pellet cellular debris and nuclei. Layer two milliliters of the supinate for each per or sucrose gradient with one gradient for each whole cortex and cap the tubes. Then centrifuge them in a fixed angle rotor at 32, 500 times gravity for five minutes at four degrees Celsius in a Beckman J 2 21 centrifuge.
Using appropriate adapters when completed, the gradient should give a strong SN band pipette off and discard the solution above the SN band. Using a glass pasta pipette pipettes off the SN band at the 15 to 23%interface, one gradient generally gives about 0.9 to 1.1 milliliters of sns. After that, transfer it to a conical tube and store on ice.
Then adjust the salt concentration of the SNS by adding one 10th volume of 10 times stimulation buffer. Optionally add 1000 times calcium chloride to give a final concentration of 12 ano molar. Next, add one millimolar TTX stock to give a final concentration of one micromolar to suppress non-specific excitation.
The next step is to use the SNS in the applicable downstream application, such as protein translation studies. For other applications, SN lysate may be cleaned up or concentrated using the Pierce SDS page sample prep kit. The protein concentration of the SNS can be determined using the micro BCA protein assay kit.
Here is an example of six bands or fractions. When the mouse cortex was homogenized and separated on discontinuous perol sucrose gradients, the enriched SNS were contained in Band five at the 23 to 15%interface, this band was removed and examined by electron microscopy. An example of intact synaptic vesicles and the preservation of pre-synaptic and post-synaptic elements are shown here in the western blot.
An increase in synaptic markers and a decrease in impurities in the SN band from a discontinuous per sucrose gradient are shown to confirm that the increase in S 35 methionine incorporation upon addition of glutamate is due to de novo protein synthesis. 40 micromolar anesa mycin a protein synthesis inhibitor was added. This figure shows the marked decrease in S 35 methionine incorporation in the presence of anso mycin with or without glutamate present when compared to the basal levels.
Thus, the discontinuous peral sucrose gradients quickly produce highly active and relatively pure SNS that can be used to study protein translation. This figure shows that band five from the discontinuous per core sucrose gradient contain the highest levels of de novo protein synthesis. The SNS prepared by passing homogenized cortex through a series of filters with decreasing pore size and then centrifuging over a discontinuous per core sucrose gradient for comparison contain more broken membranes and less whole SNS than SNS prepared from the discontinuous perol sucrose gradient method SNS prepared using the discontinuous peral sucrose gradient method are shown to contain more de novo protein synthesis activity then SNS prepared using the filtration method.
SNS prepared from younger mice are shown to have greater translational activity than older mice. After watching this video, you should have a good idea of how to isolate translationally active synaptic neuro zones by homogenizing mouse cortices centrifuging the homogenate in a swinging bucket rotor, centrifuging the supernatant over a per call sucrose gradient in a fixed angle rotor, and then collecting the resulting synaptic neuro zone band.
