I'm successful in separating the presynaptic and PSD fraction using a method that published in a Neuron article. I have some native gel running complication with the pre-synaptic membrane fraction as the gel goes bizarre. I found that the triton X-100 in the presynaptic fraction causing this while running native page gel. I even found the same while running in SDS/PAGE but I was successful after subjecting the presynaptic fraction to acetone precipitation. But the acetone precipitated presynaptic fractions is not compatible to resolve you native proteome as acetone disturbs the lipid bilayer thus no longer the native condition of your membrane proteins maintained. At the separation step of presynaptic and PSD fraction we use 1% triton at pH 8. During this step the presynaptic fraction is resolved in the supernatant along with 1% triton. I used to filter (used 100 K esp for running native page also used 10 K to 30 K filters) and exchange the presynaptic fraction buffer composition (ph 8 to 7.4) to get rid of triton but instead it gets concentrated to form a micelle I persume so. How do I get void this triton from this fraction without affecting the nativity of a proteome? Does anyone have any idea how do I over this situation?
Consider Blue Native Gel Electrophoresis which separates membrane protein complexes in their native form.
Actually I'm working on blue native but the problem I face is that the presynaptic fraction contains triton x-100. The presynaptic fraction is the supernant part which I get after separation. Therefore when I compare the presynaptic and the PSD fraction (is the pellet) I have to maintain the equal amount of protein (Does it make sense to compare both the presynaptic membrane fraction and the PSD fraction?). But always I find that after staining the gel the presynaptic fraction is less protein concentration than the PSD fraction despite that I get more more concentration OD value for the presynaptic fraction. How far dialysis would help me that I don't want to remove completely triton x-100 from the presynaptic fraction as I need to run blue native and also I need to compare it with the PSD fraction in which case we need to extract (extraction buffer will contain the detergent) the proteins from the pellet. But the Amicon filtration doesn't help me.
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