Separations with Millipore and Pall Membranes
In anticipation of the pnc-Si separation paper, I have performed a couple of preliminary separations using NanoSeps and MicroCons. I applied 200ul of 1:20 brain extract ( ~.9mg/mL) to 100k NanoSeps and MicroCons, and I spun the samples for 3 minutes at 14000g. I had wanted to collect the retentate as is from the top, but they had both almost run dry (maybe 10uL left in each case). I pipetted 50uL of PBS on the tops, agitated, and removed 50uL. I removed the filtrate as is. I ran all samples on a gel and silver stained.
From this gel we can see an obvious cutoff somewhere under 100k. Importantly we do not see reduction of small species in the retentate. Remember that this was mostly spun dry and all these small proteins are remaining behind. I have never been able to spin a SepCon dry, but it’s something I will be trying. We hypothesize that we will see more of the small species pass like we do in diffusion. This is probably a reasonable comparison because I will be trying to separation protein concentrations around 1mg/mL in the SepCons, and the 100k cutoff fall around our range.
S=standards, C=control, R=retentate, F=filtrate