I'm currently trying to purify Outer Membrane Vesicles (OMVs) following their extraction from the bacteria I'm working with. After the initial extraction the concentration of protein seems good (mixture of OMVs, free protein, flagella, etc) with ~600ug/ml (from a BCA assay) of protein being stored in Tris-HCL. To purify the OMVs I've been passing them through a sucrose gradient. I've based the protocol for this on these two papers: Article Methods of isolation and purification of outer membrane vesi...
Article Flagella proteins contribute to the production of outer memb...
In short, I've been layering 1ml layers of sucrose solution at different concentrations (2.5M, 1.6M and 0.6M) and then layering 100ul of my extracted OMVs on top. This is then centrifuged at 200,000g for 16hrs at 4C. Fractions are then taken from these gradients and diluted in PBS before being centrifuged at 150,000g for 3hrs to pellet the OMVs/other proteins and resuspend them in Tris-HCL.
However, so far when performing the BCA assay to check the OMVs are in the new solution, the concentrations are 0. This is true for all fractions taken from the column and after resuspending any potential pellet formed at the bottom of the column. I've tried adjusting the time and force the time and force the column is centrifuged for but that doesn't seem to make a difference.
Does anyone happen to have any experience regarding sucrose density gradients, or can possibly see something I'm missing? I'm at a bit of a loss as to what to do about this other then order a different medium (Optiprep) to form the density gradient and give that a go.
Any comments or suggestions would be very much appreciated : )
Hi Peter Gallagher - I have quite extensive experience with a variety of density gradients, including both sucrose and optiprep (i.e., iodixanol).
I'm happy to discuss in more detail if you'd like to message me, but the first thought is if your vesicles have too low of a density to even push through the first layer of sucrose.
Ross VerHeul, I have a few questions of my own regarding the same procedure; how may I reach you?
Hi Ross VerHeul, thank you for your answer. Turns out since asking this question the issue wasn't the with the purification.
During the initial extraction there were issues with using too little culture, and the OMVs possibly aggregating in the filter. The initial reading from the BCA I was getting was likely caused by residual LB broth being present in the resuspended sample.
I'm still trying to get the initial extraction right before moving on the purification but I will bare your answer in mind when I get to that step. Thanks again :D
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