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I am reading a journal paper where they analyse the proteome of synaptosomes. In this paper, they isolate synaptosomes from the hippocampi of mice. I know that synaptosomes contain the complete presynaptic terminal along with the postsynaptic membrane and post-synaptic density.

I am trying to understand the subcellular fractionation protocol they used for isolating the synaptosomes in the materials and methods section.

I know that synaptosomes are isolated from the subcellular fractionation of homogenised tissue. What I don't understand is what is meant by the 'crude membrane fraction P2'. I am wondering what exactly this crude membrane fraction P2 is because it is from this fraction that synaptosomes are isolated (via sucrose density gradient centrifugation).

I have read other papers where they also isolated synaptosomes. However in these papers they also mention that they homogenise brain tissue, and isolate the synaptosomes from the crude membrane fraction P2.

Any advice is appreciated.

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The first google result I get for the search term "crude membrane fraction P2" is a paper from 2014 with the title Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient. It includes a step-by-step protocol; I'll post the most relevant steps here:

  1. Low Speed Centrifugation to Remove the Nuclear Fraction (Yields Supernatant S1)

Centrifuge the homogenate in a fixed angle rotor at 900 x g for 10 min at 4 °C. Transfer the supernatant (S1) to a new, labeled 13 ml tube and resuspend the nuclear fraction pellet (P1) in 500 μl of 0.32 M HEPES-buffered sucrose. NOTE: The (P1) fraction can be stored at -80 °C and used for western blot analysis of the nuclear fraction.

  1. Enrichment of the Crude Synaptosomal Fraction (Yields Pellet P2)

Centrifuge the supernatant (S1) at 10,000 x g for 15 min at 4 °C. Remove the supernatant (S2) and reserve 500 μl in a microcentrifuge tube to store at -80 °C for subsequent protein quantification and western blot analysis of the cytosolic/light membrane fraction. Resuspend the remaining crude synaptosomal fraction pellet (P2) in 1 ml of 0.32 M HEPES-buffered sucrose solution and then add another 3 ml of 0.32 M HEPES-buffered sucrose solution.

Centrifuge at 10,000 x g for 15 min at 4 °C. Remove the supernatant (S2’) and reserve 500 μl in a microcentrifuge tube to store at -80 °C for subsequent protein quantification and western blot analysis of the cytosolic/light membrane fraction. Save the washed crude synaptosomal pellet (P2’) in the polypropylene tube.

So S and P refer to the supernatant and pellet after centrifuging, respectively. P1 and S1 are from the first round of centrifugation; the pellet P1 contains the cell nucleus and the supernatant S1 goes on to the next steps. The second round of centrifuging gives P2 and S2. P2 contains the synaptosomes, but also other membranous material which will need to be removed as S3 in later steps.

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