Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

The overall goal of this procedure is to demonstrate subcellular fractionation of brain tissue using a discontinuous sucrose gradient to enrich for proteins from the synaptic membranes and posts synaptic densities. This is accomplished by first homogenizing the brain tissue with a powered homogenizer. Next are a series of centrifugation steps to prepare crude membrane protein extracts.

The membrane extracts are then layered on a discontinuous sucrose gradient for ultracentrifugation. The final step is to collect the proteins from the gradient. Ultimately, the results can show changes in synaptic protein levels through Western blot analysis.

This is a well-established technique that's been used by neuroscientists for over 40 years. We use it in our laboratory to understand how the protein composition of the synapse changes when we perturb different Neurotransmitter systems. The main advantage of this technique over other methods, like per call gradients or continuous sucrose gradients, is that discontinuous sucrose gradients are easier to build by hand.

This protocol makes use of a motor driven glass Teflon Homogenizer always replace the pestle between samples and rinse and dry. The homogenizer first, load the dissected tissue into a 13 milliliter tube with four milliliters of 0.32 molar HEPA buffered sucrose solution. Then transfer the tissue to a clean glass Homogenizer.

Set the motor to 900 RPM and homogenize the sample with 12 strokes over 30 seconds using less than four grams of tissue ensures a good fractionation transfer the sample back to the tube and store a 100 microliter aliquot at negative 80 degrees Celsius for protein analysis. Next, separate out the nuclear material by spinning the homogenate at 900 G for 10 minutes. At four degrees Celsius, transfer the supernatant to a new 15 milliliter tube.

The supernatant contains the crude membrane fraction. Now to enrich for the crude synaptosome centrifuge, the supernatant at 10, 000 G for 15 minutes at four degrees Celsius, remove the supernatant. The pellet contains the crude synaptosome fraction Resus.

Suspend it in one milliliter of 0.32 molar sucrose solution. Then at another three milliliters of solution to the suspension centrifuge the crude fraction for 15 minutes At 10, 000 G at four degrees Celsius, remove the supernatant. Now quickly lice the pellet, resuspend the pellet in a milliliter of pure water.

Then add another three milliliters of water rapidly. Transfer this to the homogenizer. Apply three strokes by hand and transfer it back to the 13 milliliter tube.

Quickly adjust the sample back to four millimolar HEAs with an addition of 16 microliters of one molar HEAs and mix it by inversion. Now put the sample on a rotator at four degrees Celsius for half an hour. To ensure the sample lysis, finish the fractionation by pelleting the Lysed P two prime fraction.

Spin it down at 25, 000 G for 20 minutes at four degrees Celsius, remove the supernatant, resuspend the pellet in a milliliter of sucrose solution. This contains the enriched synap episomal membranes. First, pre-measure the buffers to make a discontinuous sucrose gradient.

Load three tubes as follows, one with exactly 3.5 milliliters of 1.2 molar heaps buffered sucrose solution. Another with exactly three milliliters of 1.0 molar solution, and the third with three milliliters of 0.8 molar solution. Next, with a glass pester pipette, transfer the 1.2 molar sucrose solution to a 12 milliliter polymer ultracentrifuge tube.

With a new pipette, carefully layer the 1.0 molar solution onto the 1.2 molar solution. Do not disturb the 1.2 molar solution or introduce bubbles with the pipette. Use the same technique to layer the 0.8 molar solution on top.

Take caution. Small vibrations from a bench vortex or micro centrifuge are enough to disturb the integrity of the gradient. It's important to build the gradients carefully and efficiently.

If it takes too long or if the gradient is exposed to vibration, you won't be able to recover the synaptic membranes in the later steps. Now with another pastor pipette layer the enriched synap episomal membrane fraction on top of the prepared gradient. Precisely balance the gradient in an ultracentrifuge in a swinging bucket rotor.

Then spin it down at 150, 000 G for two hours at four degrees Celsius. Be very careful when removing the gradient from the bucket after the centrifugation, there should be clear bands of sucrose solution. With protein fractions at each interface and a pellet, the fractions should not be diffuse.

Using an 18 gauge needle and a one milliliter syringe, puncture the lowest part of the to 1.2 molar interface and withdraw the white band of protein, which contains the synaptic plasma membranes. Record the volume of this layer. Transfer the layer to a 3.5 milliliter thick walled ultracentrifuge tube.

Add exactly 2.5 volumes of four millimolar HEAs. Then prepare a balancer tube with 0.32 molar HEAs buffered sucrose solution in a fixed angle rotor, ultracentrifuge the sample at 200, 000 G for 30 minutes at four degrees Celsius. Then discard the Senna and resuspend the synaptic plasma membrane pellet.

In 300 microliters of 50 millimolar HEAs two millimolar EDTA solution. The post-synaptic density fraction or PSD fraction can now be collected by further use of the same techniques. The steps are clearly outlined in the text protocol.

A western blot was run with a total protein, the synaptic plasma membrane, and first post-synaptic density protein fractions. The PSD fraction underwent just one detergent treatment, and the tissue was from mouse striatum. Presynaptic proteins like the dopamine transporter are enriched in the SPM fraction, but are eliminated in the subsequent detergent step for PSD enrichment.

While attempting this procedure, it's important to remember to keep all samples on ice and to include protease and phosphatase inhibitors Following this procedure. Other methods like western blood analysis or two dimensional gel electrophoresis can be performed. Then you can answer questions like how drug treatment affects receptor levels at the synapse.